Microfluidic devices and methods of use

ABSTRACT

A microfluidic device comprises pumps, valves, and fluid oscillation dampers. In a device employed for sorting, an entity is flowed by the pump along a flow channel through a detection region to a junction. Based upon an identity of the entity determined in the detection region, a waste or collection valve located on opposite branches of the flow channel at the junction are actuated, thereby routing the entity to either a waste pool or a collection pool. A damper structure may be located between the pump and the junction. The damper reduces the amplitude of oscillation pressure in the flow channel due to operation of the pump, thereby lessening oscillation in velocity of the entity during sorting process. The microfluidic device may be formed in a block of elastomer material, with thin membranes of the elastomer material deflectable into the flow channel to provide pump or valve functionality.

CROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] The instant nonprovisional patent application claims priorityfrom U.S. Provisional Patent Application No. 60/237,938, entitledMICROFLUIDIC DEVICES AND METHODS OF USE, filed Oct. 3, 2000. The instantnonprovisional patent application also claims priority from U.S.Provisional Patent Application No. 60/237,937, entitled VELOCITYINDEPENDENT MICROFLUIDIC FLOW CYTOMETRY, filed Oct. 3, 2000. Theseprovisional patent applications are incorporated by reference herein forall purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] Work described herein has been supported, in part, by NationalInstitute of Health grant HG-01642-02. The United States Government maytherefore have certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Sorting of objects based upon size is extremely useful in manytechnical fields. For example, many assays in biology requiredetermination of the size of molecular-sized entities. Of particularimportance is the measurement of length distribution of DNA molecules ina heterogeneous solution. This is commonly done using gelelectrophoresis, in which the molecules are separated by their differingmobility in a gel matrix in an applied electric field, and theirpositions detected by absorption or emission of radiation. The lengthsof the DNA molecules are then inferred from their mobility.

[0004] While powerful, electrophoretic methods pose disadvantages. Formedium to large DNA molecules, resolution, i.e. the minimum lengthdifference at which different molecular lengths may be distinguished, islimited to approximately 10% of the total length. For extremely largeDNA molecules, the conventional sorting procedure is not workable.Moreover, gel electrophoresis is a relatively lengthy procedure, and mayrequire on the order of hours or days to perform.

[0005] The sorting of cellular-sized entities is also an important task.Conventional flow cell sorters are designed to have a flow chamber witha nozzle and are based on the principle of hydrodynamic focusing withsheath flow. Most conventional cell sorters combine the technology ofpiezo-electric drop generation and electrostatic deflection to achievedroplet generation and high sorting rates. However, this approach offerssome important disadvantages. One disadvantage is that the complexity,size, and expense of the sorting device requires that it be reusable inorder to be cost-effective. Reuse can in turn lead to problems withresidual materials causing contamination of samples and turbulent fluidflow.

[0006] Another disadvantage of conventional sorting technologies is aninability to readily integrate sorting with other activities. Forexample, due to the mechanical complexity of conventional sortingapparatuses, pre-sorting activities such as labeling and post-sortingactivities such as crystallization are typically performed on differentdevices, requiring physical transfer of the pre- and post-sorted sampleto the sampling apparatus.

[0007] This transfer requires precise and careful handling in order toprevent any loss of the frequently small volumes of sample involved.Moreover, sample handling for conventional sorter structures istime-consuming. The resulting delay may hinder analysis of materialshaving limited lifetimes, or prevent sorting that is based upontime-dependent criteria.

[0008] Therefore, there is a need in the art for a simple, inexpensive,and easily fabricated integratable sorting device which relies upon themechanical control of fluid flow rather than upon electrical interactionbetween the particle and the solute.

SUMMARY OF THE INVENTION

[0009] An embodiment of a microfluidic device in accordance with thepresent invention comprises pumps, valves, and fluid oscillationdampers. In a device employed for sorting, an entity is flowed by thepump along a flow channel through a detection region to a junction.Based upon an identity of the entity determined in the detection region,a waste or collection valve located on opposite branches of the flowchannel is actuated, thereby routing the entity to either a waste poolor a collection pool. A damper structure may be located between the pumpand the junction. The damper reduces the amplitude of oscillationpressure in the flow channel due to operation of the pump, therebylessening oscillation in velocity of the entity during sorting process.The microfluidic device may be formed in a block of elastomer material,with thin membranes of the elastomer material deflectable into the flowchannel to provide pump or valve functionality.

[0010] An embodiment of a microfluidic device in accordance with thepresent invention comprises a flow channel, a pump operativelyinterconnected to said flow channel for moving a fluid in said flowchannel, and a damper operatively interconnected to said flow channelfor reducing the fluid oscillation in said flow channel.

[0011] An embodiment of a microfluidic sorting device comprises a firstflow channel formed in a first layer of elastomer material, a first endof the first flow channel in fluid communication with a collection pooland a second end of the first flow channel in fluid communication with awaste pool. A second flow channel is formed in the first elastomerlayer, a first end of the second flow channel in fluid communicationwith an injection pool and a second end of the second flow channel influid communication with the first flow channel at a junction. Acollection valve is adjacent to a first side of the junction proximateto the collection pool, the collection valve comprising a first recessformed in a second elastomer layer overlying the first elastomer layer,the first recess separated from the first flow channel by a firstmembrane portion of the second elastomer layer deflectable into thefirst flow channel. A waste valve is adjacent to a second side of thejunction proximate to the waste pool, the waste valve comprising asecond recess formed in the second elastomer layer separated from thesecond flow channel by a second membrane portion of the second elastomerlayer deflectable into the first flow channel. A pump adjacent to athird side of the junction proximate to the injection pool, the pumpcomprising at least three pressure channels formed in the secondelastomer layer and separated from second flow channel by third membraneportions of the second elastomer layer deflectable into the second flowchannel. A detection region is positioned between the injection pool andthe junction, one of an open and closed state of the collection valveand the waste valve determined by an identity of a sortable entitydetected in the detection region.

[0012] An embodiment of a damper in accordance with the presentinvention for a microfluidic device comprises a flow channel formed inan elastomer material, and an energy absorber adjacent to the flowchannel and configured to absorb an energy of oscillation of a fluidpositioned within the flow channel.

[0013] An embodiment of a sorting method in accordance with the presentinvention comprises deflecting a first elastomer membrane of anelastomer block into a flow channel to cause a sortable entity to flowinto a detection region positioned upstream of a junction in the flowchannel. The detection region is interrogated to identify the sortableentity within the detection region. Based upon an identity of thesortable entity, one of a second membrane and a third membrane of theelastomer block are deflected into one of a first branch flow channelportion and a second branch flow channel portion respectively, locateddownstream of the junction. This causes the sortable entity to flow toone of a collection pool or a waste pool.

[0014] An embodiment of a method for dampening pressure oscillations ina flow channel comprises providing an energy absorber adjacent to theflow channel, such that the energy absorber experiences a change inresponse the pressure oscillations.

[0015] These and other embodiments of the present invention, as well asits advantages and features, are described in more detail in conjunctionwith the text below and attached figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is an illustration of a first elastomeric layer formed ontop of a micromachined mold.

[0017]FIG. 2 is an illustration of a second elastomeric layer formed ontop of a micromachined mold.

[0018]FIG. 3 is an illustration of the elastomeric layer of FIG. 2removed from the micromachined mold and positioned over the top of theelastomeric layer of FIG. 1

[0019]FIG. 4 is an illustration corresponding to FIG. 3, but showing thesecond elastomeric layer positioned on top of the first elastomericlayer.

[0020]FIG. 5 is an illustration corresponding to FIG. 4, but showing thefirst and second elastomeric layers bonded together.

[0021]FIG. 6 is an illustration corresponding to FIG. 5, but showing thefirst micromachined mold removed and a planar substrate positioned inits place.

[0022]FIG. 7A is an illustration corresponding to FIG. 6, but showingthe elastomeric structure sealed onto the planar substrate.

[0023]FIGS. 7B is a front sectional view corresponding to FIG. 7A,showing an open flow channel.

[0024] FIGS. 7C-7G are illustrations showing steps of a method forforming an elastomeric structure having a membrane formed from aseparate elastomeric layer.

[0025]FIG. 8A and 8B illustrates valve opening vs. applied pressure forvarious flow channels.

[0026]FIG. 9 illustrates time response of a 100 μm×100 μm×10 μm RTVmicrovalve.

[0027]FIG. 12A is a top schematic view of an on/off valve.

[0028]FIG. 12B is a sectional elevation view along line 23B-23B in FIG.12A

[0029]FIG. 13A is a top schematic view of a peristaltic pumping system.

[0030]FIG. 13B is a sectional elevation view along line 24B-24B in FIG.13A

[0031]FIG. 14 is a graph showing experimentally achieved pumping ratesvs. frequency for an embodiment of the peristaltic pumping system ofFIG. 13.

[0032]FIG. 15A is a top schematic view of one control line actuatingmultiple flow lines simultaneously.

[0033]FIG. 15B is a sectional elevation view along line 26B-26B in FIG.15A

[0034]FIG. 16 is a schematic illustration of a multiplexed systemadapted to permit flow through various channels.

[0035]FIG. 17A is a plan view of a flow layer of an addressable reactionchamber structure.

[0036]FIG. 17B is a bottom plan view of a control channel layer of anaddressable reaction chamber structure.

[0037]FIG. 17C is an exploded perspective view of the addressablereaction chamber structure formed by bonding the control channel layerof FIG. 17B to the top of the flow layer of FIG. 17A.

[0038]FIG. 17D is a sectional elevation view corresponding to FIG. 17C,taken along line 28D-28D in FIG. 17C.

[0039]FIG. 18 is a schematic of a system adapted to selectively directfluid flow into any of an array of reaction wells.

[0040]FIG. 19 is a schematic of a system adapted for selectable lateralflow between parallel flow channels.

[0041]FIG. 20A is a bottom plan view of first layer (i.e.: the flowchannel layer) of elastomer of a switchable flow array.

[0042]FIG. 20B is a bottom plan view of a control channel layer of aswitchable flow array.

[0043]FIG. 20C shows the alignment of the first layer of elastomer ofFIG. 20A with one set of control channels in the second layer ofelastomer of FIG. 20B.

[0044]FIG. 20D also shows the alignment of the first layer of elastomerof FIG. 20A with the other set of control channels in the second layerof elastomer of FIG. 20B.

[0045] FIGS. 21A-21J show views of one embodiment of a normally-closedvalve structure in accordance with the present invention.

[0046]FIGS. 22A and 22B show plan views illustrating operation of oneembodiment of a side-actuated valve structure in accordance with thepresent invention.

[0047]FIG. 23 shows a cross-sectional view of one embodiment of acomposite structure in accordance with the present invention.

[0048]FIG. 24 shows a cross-sectional view of another embodiment of acomposite structure in accordance with the present invention.

[0049]FIG. 25 shows a cross-sectional view of another embodiment of acomposite structure in accordance with the present invention.

[0050]FIG. 26 is a cross-sectional view of one embodiment of a damperstructure in accordance with the present invention.

[0051]FIG. 27 is a cross-sectional view of an alternative embodiment ofa damper structure in accordance with the present invention.

[0052]FIG. 28 is a plan view of another alternative embodiment of adamper structure in accordance with the present invention.

[0053] FIGS. 29A-B are cross-sectional views of operation of oneembodiment of a circulation apparatus including a damper structure inaccordance with the present invention.

[0054]FIG. 30A is a schematic plan view of a cell sorter structure inaccordance with one embodiment of the present invention.

[0055]FIG. 30B is a photograph of a plan view of the cell sorter shownin FIG. 30A.

[0056]FIGS. 31A and 31B are schematic views of a T-junction of a sorterstructure in accordance with an embodiment of the present inventionengaged in reversible sorting.

[0057]FIG. 32A plots cell velocity versus pump frequency for oneembodiment of a cell sorter in accordance with the present invention.

[0058]FIG. 32B plots mean reversible time versus pump frequency for theembodiment of a cell sorter of FIG. 32A.

[0059]FIG. 33A plots optical intensity over time for the cell sorterstructure of FIG. 33C operated at first frequency.

[0060]FIG. 33B plots optical intensity over time for the cell sorterstructure of FIG. 33C operated at first frequency.

[0061]FIG. 33C is a schematic view of a cell sorter structure inaccordance with an alternative embodiment in accordance with the presentinvention.

[0062]FIG. 34 plots flow velocity versus pump frequency for cell sortersfabricated from different elastomeric materials.

DESCRIPTION OF SPECIFIC EMBODIMENTS

[0063] I. Microfabrication Overview

[0064] The following discussion relates to formation of microfabricatedfluidic devices utilizing elastomer materials, as described generally inU.S. patent application Ser. Nos. 09/826,585 filed Apr. 6, 2001,09/724,784 filed Nov. 28, 2000, and 09/605,520, filed Jun. 27, 2000.These patent applications are hereby incorporated by reference.

[0065] 1. Methods of Fabricating

[0066] Exemplary methods of fabricating the present invention areprovided herein. It is to be understood that the present invention isnot limited to fabrication by one or the other of these methods. Rather,other suitable methods of fabricating the present microstructures,including modifying the present methods, are also contemplated.

[0067] FIGS. 1 to 7B illustrate sequential steps of a first preferredmethod of fabricating the present microstructure, (which may be used asa pump or valve). FIGS. 8 to 18 illustrate sequential steps of a secondpreferred method of fabricating the present microstructure, (which alsomay be used as a pump or valve).

[0068] As will be explained, the preferred method of FIGS. 1 to 7Binvolves using pre-cured elastomer layers which are assembled andbonded. In an alternative method, each layer of elastomer may be cured“in place”. In the following description “channel” refers to a recess inthe elastomeric structure which can contain a flow of fluid or gas.

[0069] Referring to FIG. 1, a first micro-machined mold 10 is provided.Micro-machined mold 10 may be fabricated by a number of conventionalsilicon processing methods, including but not limited tophotolithography, ion-milling, and electron beam lithography.

[0070] As can be seen, micro-machined mold 10 has a raised line orprotrusion 11 extending therealong. A first elastomeric layer 20 is caston top of mold 10 such that a first recess 21 will be formed in thebottom surface of elastomeric layer 20, (recess 21 corresponding indimension to protrusion 11), as shown.

[0071] As can be seen in FIG. 2, a second micro-machined mold 12 havinga raised protrusion 13 extending therealong is also provided. A secondelastomeric layer 22 is cast on top of mold 12, as shown, such that arecess 23 will be formed in its bottom surface corresponding to thedimensions of protrusion 13.

[0072] As can be seen in the sequential steps illustrated in FIGS. 3 and4, second elastomeric layer 22 is then removed from mold 12 and placedon top of first elastomeric layer 20. As can be seen, recess 23extending along the bottom surface of second elastomeric layer 22 willform a flow channel 32.

[0073] Referring to FIG. 5, the separate first and second elastomericlayers 20 and 22 (FIG. 4) are then bonded together to form an integrated(i.e.: monolithic) elastomeric structure 24.

[0074] As can been seen in the sequential step of FIGS. 6 and 7A,elastomeric structure 24 is then removed from mold 10 and positioned ontop of a planar substrate 14. As can be seen in FIG. 7A and 7B, whenelastomeric structure 24 has been sealed at its bottom surface to planarsubstrate 14, recess 21 will form a flow channel 30.

[0075] The present elastomeric structures form a reversible hermeticseal with nearly any smooth planar substrate. An advantage to forming aseal this way is that the elastomeric structures may be peeled up,washed, and re-used. In preferred aspects, planar substrate 14 is glass.A further advantage of using glass is that glass is transparent,allowing optical interrogation of elastomer channels and reservoirs.Alternatively, the elastomeric structure may be bonded onto a flatelastomer layer by the same method as described above, forming apermanent and high-strength bond. This may prove advantageous whenhigher back pressures are used.

[0076] As can be seen in FIG. 7A and 7B, flow channels 30 and 32 arepreferably disposed at an angle to one another with a small membrane 25of substrate 24 separating the top of flow channel 30 from the bottom offlow channel 32.

[0077] In preferred aspects, planar substrate 14 is glass. An advantageof using glass is that the present elastomeric structures may be peeledup, washed and reused. A further advantage of using glass is thatoptical sensing may be employed. Alternatively, planar substrate 14 maybe an elastomer itself, which may prove advantageous when higher backpressures are used.

[0078] The method of fabrication just described may be varied to form astructure having a membrane composed of an elastomeric materialdifferent than that forming the walls of the channels of the device.This variant fabrication method is illustrated in FIGS. 7C-7G.

[0079] Referring to FIG. 7C, a first micro-machined mold 10 is provided.Micro-machined mold 10 has a raised line or protrusion 11 extendingtherealong. In FIG. 7D, first elastomeric layer 20 is cast on top offirst micro-machined mold 10 such that the top of the first elastomericlayer 20 is flush with the top of raised line or protrusion 11. This maybe accomplished by carefully controlling the volume of elastomericmaterial spun onto mold 10 relative to the known height of raised line11. Alternatively, the desired shape could be formed by injectionmolding.

[0080] In FIG. 7E, second micro-machined mold 12 having a raisedprotrusion 13 extending therealong is also provided. Second elastomericlayer 22 is cast on top of second mold 12 as shown, such that recess 23is formed in its bottom surface corresponding to the dimensions ofprotrusion 13.

[0081] In FIG. 7F, second elastomeric layer 22 is removed from mold 12and placed on top of third elastomeric layer 222. Second elastomericlayer 22 is bonded to third elastomeric layer 20 to form integralelastomeric block 224 using techniques described in detail below. Atthis point in the process, recess 23 formerly occupied by raised line 13will form flow channel 23.

[0082] In FIG. 7G, elastomeric block 224 is placed on top of firstmicro-machined mold 10 and first elastomeric layer 20. Elastomeric blockand first elastomeric layer 20 are then bonded together to form anintegrated (i.e.: monolithic) elastomeric structure 24 having a membranecomposed of a separate elastomeric layer 222.

[0083] When elastomeric structure 24 has been sealed at its bottomsurface to a planar substrate in the manner described above inconnection with FIG. 7A, the recess formerly occupied by raised line 11will form flow channel 30.

[0084] The variant fabrication method illustrated above in conjunctionwith FIGS. 7C-7G offers the advantage of permitting the membrane portionto be composed of a separate material than the elastomeric material ofthe remainder of the structure. This is important because the thicknessand elastic properties of the membrane play a key role in operation ofthe device. Moreover, this method allows the separate elastomer layer toreadily be subjected to conditioning prior to incorporation into theelastomer structure. As discussed in detail below, examples ofpotentially desirable condition include the introduction of magnetic orelectrically conducting species to permit actuation of the membrane,and/or the introduction of dopant into the membrane in order to alterits elasticity.

[0085] While the above method is illustrated in connection with formingvarious shaped elastomeric layers formed by replication molding on topof a micromachined mold, the present invention is not limited to thistechnique. Other techniques could be employed to form the individuallayers of shaped elastomeric material that are to be bonded together.For example, a shaped layer of elastomeric material could be formed bylaser cutting or injection molding, or by methods utilizing chemicaletching and/or sacrificial materials as discussed below in conjunctionwith the second exemplary method.

[0086] An alternative method fabricates a patterned elastomer structureutilizing development of photoresist encapsulated within elastomermaterial. However, the methods in accordance with the present inventionare not limited to utilizing photoresist. Other materials such as metalscould also serve as sacrificial materials to be removed selective to thesurrounding elastomer material, and the method would remain within thescope of the present invention. For example, gold metal may be etchedselective to RTV 615 elastomer utilizing the appropriate chemicalmixture.

[0087] 2. Layer and Channel Dimensions

[0088] Microfabricated refers to the size of features of an elastomericstructure fabricated in accordance with an embodiment of the presentinvention. In general, variation in at least one dimension ofmicrofabricated structures is controlled to the micron level, with atleast one dimension being microscopic (i.e. below 1000 μm).Microfabrication typically involves semiconductor or MEMS fabricationtechniques such as photolithography and spincoating that are designedfor to produce feature dimensions on the microscopic level, with atleast some of the dimension of the microfabricated structure requiring amicroscope to reasonably resolve/image the structure.

[0089] In preferred aspects, flow channels 30, 32, 60 and 62 preferablyhave width-to-depth ratios of about 10:1. A non-exclusive list of otherranges of width-to-depth ratios in accordance with embodiments of thepresent invention is 0.1:1 to 100:1, more preferably 1:1 to 50:1, morepreferably 2:1 to 20:1, and most preferably 3:1 to 15:1. In an exemplaryaspect, flow channels 30, 32, 60 and 62 have widths of about 1 to 1000microns. A non-exclusive list of other ranges of widths of flow channelsin accordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 1000 microns, more preferably 0.2 to500 microns, more preferably 1 to 250 microns, and most preferably 10 to200 microns. Exemplary channel widths include 0.1 μm, 1 μm, 2 μm, 5 μm,10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm,110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm,200 μm, 210 μm, 220 μm, 230 μm, 240 μm, and 250 μm.

[0090] Flow channels 30, 32, 60, and 62 have depths of about 1 to 100microns. A non-exclusive list of other ranges of depths of flow channelsin accordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 500 microns, more preferably 0.2 to 250microns, and more preferably 1 to 100 microns, more preferably 2 to 20microns, and most preferably 5 to 10 microns. Exemplary channel depthsinclude including 0.01 μm, 0.02 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.5 μm, 1μm, 2 μm, 3 μm, 4 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm, 15 μm, 17.5 μm, 20μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm,and 250 μm.

[0091] The flow channels are not limited to these specific dimensionranges and examples given above, and may vary in width in order toaffect the magnitude of force required to deflect the membrane asdiscussed at length below in conjunction with FIG. 27. For example,extremely narrow flow channels having a width on the order of 0.01 μmmay be useful in optical and other applications, as discussed in detailbelow. Elastomeric structures which include portions having channels ofeven greater width than described above are also contemplated by thepresent invention, and examples of applications of utilizing such widerflow channels include fluid reservoir and mixing channel structures.

[0092] The Elastomeric layers may be cast thick for mechanicalstability. In an exemplary embodiment, elastomeric layer 22 of FIG. 1 is50 microns to several centimeters thick, and more preferablyapproximately 4 mm thick. A non-exclusive list of ranges of thickness ofthe elastomer layer in accordance with other embodiments of the presentinvention is between about 0.1 micron to 10 cm, 1 micron to 5 cm, 10microns to 2 cm, 100 microns to 10 mm.

[0093] Accordingly, membrane 25 of FIG. 7B separating flow channels 30and 32 has a typical thickness of between about 0.01 and 1000 microns,more preferably 0.05 to 500 microns, more preferably 0.2 to 250, morepreferably 1 to 100 microns, more preferably 2 to 50 microns, and mostpreferably 5 to 40 microns. As such, the thickness of elastomeric layer22 is about 100 times the thickness of elastomeric layer 20. Exemplarymembrane thicknesses include 0.01 μm, 0.02 μm, 0.03 μm, 0.05 μm, 0.1 μm,0.2 μm, 0.3 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm,15 μm, 17.5 μm, 20 μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100μm, 150 μm, 200 μm, 250 μm, 300 μm, 400 μm, 500 μm, 750 μm, and 1000 μm.

[0094] 3. Soft Lithographic Bonding

[0095] Preferably, elastomeric layers are bonded together chemically,using chemistry that is intrinsic to the polymers comprising thepatterned elastomer layers. Most preferably, the bonding comprises twocomponent “addition cure” bonding.

[0096] In a preferred aspect, the various layers of elastomer are boundtogether in a heterogenous bonding in which the layers have a differentchemistry. Alternatively, a homogenous bonding may be used in which alllayers would be of the same chemistry. Thirdly, the respective elastomerlayers may optionally be glued together by an adhesive instead. In afourth aspect, the elastomeric layers may be thermoset elastomers bondedtogether by heating.

[0097] In one aspect of homogeneous bonding, the elastomeric layers arecomposed of the same elastomer material, with the same chemical entityin one layer reacting with the same chemical entity in the other layerto bond the layers together. In one embodiment, bonding between polymerchains of like elastomer layers may result from activation of acrosslinking agent due to light, heat, or chemical reaction with aseparate chemical species.

[0098] Alternatively in a heterogeneous aspect, the elastomeric layersare composed of different elastomeric materials, with a first chemicalentity in one layer reacting with a second chemical entity in anotherlayer. In one exemplary heterogenous aspect, the bonding process used tobind respective elastomeric layers together may comprise bondingtogether two layers of RTV 615 silicone. RTV 615 silicone is a two-partaddition-cure silicone rubber. Part A contains vinyl groups andcatalyst; part B contains silicon hydride (Si—H) groups. Theconventional ratio for RTV 615 is 10A:1B. For bonding, one layer may bemade with 30A:1B (i.e. excess vinyl groups) and the other with 3A:1B(i.e. excess Si—H groups). Each layer is cured separately. When the twolayers are brought into contact and heated at elevated temperature, theybond irreversibly forming a monolithic elastomeric substrate.

[0099] In an exemplary aspect of the present invention, elastomericstructures are formed utilizing Dow Corning Sylgard 182, 184 or 186, oraliphatic urethane diacrylates such as (but not limited to) Ebecryl 270or Irr 245 from UCB Chemical.

[0100] In one embodiment in accordance with the present invention,two-layer elastomeric structures were fabricated from Dow Coming Sylgard184. The layer containing the flow channels had an A:B ratio of 20:1 wasspin coated at 5000 rpm. The layer containing the control channels hadan A:B ratio of 5:1.

[0101] In another embodiment in accordance with the present invention,two-layer elastomeric structures were fabricated from pure acrylatedUrethane Ebe 270. A thin bottom layer was spin coated at 8000 rpm for 15seconds at 170° C. The top and bottom layers were initially cured underultraviolet light for 10 minutes under nitrogen utilizing a Model ELC500 device manufactured by Electrolite corporation. The assembled layerswere then cured for an additional 30 minutes. Reaction was catalyzed bya 0.5% vol/vol mixture of Irgacure 500 manufactured by Ciba-GeigyChemicals. The resulting elastomeric material exhibited moderateelasticity and adhesion to glass.

[0102] In another embodiment in accordance with the present invention,two-layer elastomeric structures were fabricated from a combination of25% Ebe 270/50% Irr245/25% isopropyl alcohol for a thin bottom layer,and pure acrylated Urethane Ebe 270 as a top layer. The thin bottomlayer was initially cured for 5 min, and the top layer initially curedfor 10 minutes, under ultraviolet light under nitrogen utilizing a ModelELC 500 device manufactured by Electrolite corporation. The assembledlayers were then cured for an additional 30 minutes. Reaction wascatalyzed by a 0.5% vol/vol mixture of Irgacure 500 manufactured byCiba-Geigy Chemicals. The resulting elastomeric material exhibitedmoderate elasticity and adhered to glass.

[0103] Alternatively, other bonding methods may be used, includingactivating the elastomer surface, for example by plasma exposure, sothat the elastomer layers/substrate will bond when placed in contact.For example, one possible approach to bonding together elastomer layerscomposed of the same material is set forth by Duffy et al, “RapidPrototyping of Microfluidic Systems in Poly (dimethylsiloxane)”,Analytical Chemistry (1998), 70, 4974-4984, incorporated herein byreference. This paper discusses that exposing polydimethylsiloxane(PDMS) layers to oxygen plasma causes oxidation of the surface, withirreversible bonding occurring when the two oxidized layers are placedinto contact.

[0104] Yet another approach to bonding together successive layers ofelastomer is to utilize the adhesive properties of uncured elastomer.Specifically, a thin layer of uncured elastomer such as RTV 615 isapplied on top of a first cured elastomeric layer. Next, a second curedelastomeric layer is placed on top of the uncured elastomeric layer. Thethin middle layer of uncured elastomer is then cured to produce amonolithic elastomeric structure. Alternatively, uncured elastomer canbe applied to the bottom of a first cured elastomer layer, with thefirst cured elastomer layer placed on top of a second cured elastomerlayer. Curing the middle thin elastomer layer again results in formationof a monolithic elastomeric structure.

[0105] Bonding together of successive elastomer layers in accordancewith embodiments of the present invention need not result in amonolithic elastomeric structure. Successive layers of differentelastomer materials can be bonded together to form an embodiment of astructure in accordance with the present invention. For example GeneralElectric RTV and Dow Corning Sylgard 184 can be bonded together to forma multilayer structure.

[0106] Moreover, bonding together of successive elastomer layers inaccordance with the present invention need not be permanent. In certainembodiments, the strength of the bond between elastomer layers need onlymaintain contact and resist separation under the forces encounteredduring membrane actuation. Application of greater force will cause theelastomer layers to separate, for example allowing flushing and reuse offlow or control channels present in the separated layers.

[0107] Where encapsulation of sacrificial layers is employed tofabricate the elastomer structure, bonding of successive elastomericlayers may be accomplished by pouring uncured elastomer over apreviously cured elastomeric layer and any sacrificial materialpatterned thereupon. Bonding between elastomer layers occurs due tointerpenetration and reaction of the polymer chains of an uncuredelastomer layer with the polymer chains of a cured elastomer layer.Subsequent curing of the elastomeric layer will create a bond betweenthe elastomeric layers and create a monolithic elastomeric structure.

[0108] Referring to the first method of FIGS. 1 to 7B, first elastomericlayer 20 may be created by spin-coating an RTV mixture onmicrofabricated mold 12 at 2000 rpm's for 30 seconds yielding athickness of approximately 40 microns. Second elastomeric layer 22 maybe created by spin-coating an RTV mixture on microfabricated mold 11.Both layers 20 and 22 may be separately baked or cured at about 80° C.for 1.5 hours. The second elastomeric layer 22 may be bonded onto firstelastomeric layer 20 at about 80° C. for about 1.5 hours.

[0109] Micromachined molds 10 and 12 may be patterned photoresist onsilicon wafers. In an exemplary aspect, a Shipley SJR 5740 photoresistwas spun at 2000 rpm patterned with a high resolution transparency filmas a mask and then developed yielding an inverse channel ofapproximately 10 microns in height. When baked at approximately 200° C.for about 30 minutes, the photoresist reflows and the inverse channelsbecome rounded. In preferred aspects, the molds may be treated withtrimethylchlorosilane (TMCS) vapor for about a minute before each use inorder to prevent adhesion of silicone rubber.

[0110] 4. Suitable Elastomeric Materials

[0111] Allcock et al, Contemporary Polymer Chemistry, 2^(nd) Ed.describes elastomers in general as polymers existing at a temperaturebetween their glass transition temperature and liquefaction temperature.Elastomeric materials exhibit elastic properties because the polymerchains readily undergo torsional motion to permit uncoiling of thebackbone chains in response to a force, with the backbone chainsrecoiling to assume the prior shape in the absence of the force. Ingeneral, elastomers deform when force is applied, but then return totheir original shape when the force is removed. The elasticity exhibitedby elastomeric materials may be characterized by a Young's modulus.Elastomeric materials having a Young's modulus of between about 1 Pa-1TPa, more preferably between about 10 Pa-100 GPa, more preferablybetween about 20 Pa-1 GPa, more preferably between about 50 Pa-10 MPa,and more preferably between about 100 Pa-1 MPa are useful in accordancewith the present invention, although elastomeric materials having aYoung's modulus outside of these ranges could also be utilized dependingupon the needs of a particular application.

[0112] The systems of the present invention may be fabricated from awide variety of elastomers. In an exemplary aspect, the elastomericlayers may preferably be fabricated from silicone rubber. However, othersuitable elastomers may also be used.

[0113] In an exemplary aspect of the present invention, the presentsystems are fabricated from an elastomeric polymer such as GE RTV 615(formulation), a vinyl-silane crosslinked (type) silicone elastomer(family). However, the present systems are not limited to this oneformulation, type or even this family of polymer; rather, nearly anyelastomeric polymer is suitable. An important requirement for thepreferred method of fabrication of the present microvalves is theability to bond multiple layers of elastomers together. In the case ofmultilayer soft lithography, layers of elastomer are cured separatelyand then bonded together. This scheme requires that cured layers possesssufficient reactivity to bond together. Either the layers may be of thesame type, and are capable of bonding to themselves, or they may be oftwo different types, and are capable of bonding to each other. Otherpossibilities include the use an adhesive between layers and the use ofthermoset elastomers.

[0114] Given the tremendous diversity of polymer chemistries,precursors, synthetic methods, reaction conditions, and potentialadditives, there are a huge number of possible elastomer systems thatcould be used to make monolithic elastomeric microvalves and pumps.Variations in the materials used will most likely be driven by the needfor particular material properties, i.e. solvent resistance, stiffness,gas permeability, or temperature stability.

[0115] For example, a diluent may be included during formation of theelastomer material to alter its properties. In one embodiment, a diluentis added to the elastomer comprising the membrane layer to lessen thestiffness of the membrane and thereby reduce the actuation forcerequired. Two examples of diluent for elastomer materials are GeneralElectric SF96, and DMV-V21 manufactured by Gelest, Inc. of Tullytown,Pa. In general, the diluent is mixed with the elastomer at a ratio ofbetween about 15% and 30%.

[0116] There are many, many types of elastomeric polymers. A briefdescription of the most common classes of elastomers is presented here,with the intent of showing that even with relatively “standard”polymers, many possibilities for bonding exist. Common elastomericpolymers include polyisoprene, polybutadiene, polychloroprene,polyisobutylene, poly(styrene-butadiene-styrene), the polyurethanes, andsilicones.

[0117] Polyisoprene, polybutadiene, polychloroprene

[0118] Polyisoprene, polybutadiene, and polychloroprene are allpolymerized from diene monomers, and therefore have one double bond permonomer when polymerized. This double bond allows the polymers to beconverted to elastomers by vulcanization (essentially, sulfur is used toform crosslinks between the double bonds by heating). This would easilyallow homogeneous multilayer soft lithography by incompletevulcanization of the layers to be bonded; photoresist encapsulationwould be possible by a similar mechanism.

[0119] Polyisobutylene

[0120] Pure polyisobutylene has no double bonds, but is crosslinked touse as an elastomer by including a small amount (˜1%) of isoprene in thepolymerization. The isoprene monomers give pendant double bonds on thepolyisobutylene backbone, which may then be vulcanized as above.

[0121] Poly(styrene-butadiene-styrene)

[0122] Poly(styrene-butadiene-styrene) is produced by living anionicpolymerization (that is, there is no natural chain-terminating step inthe reaction), so “live” polymer ends can exist in the cured polymer.This makes it a natural candidate for the present photoresistencapsulation system (where there will be plenty of unreacted monomer inthe liquid layer poured on top of the cured layer). Incomplete curingwould allow homogeneous multilayer soft lithography (A to A bonding).The chemistry also facilitates making one layer with extra butadiene(“A”) and coupling agent and the other layer (“B”) with a butadienedeficit (for heterogeneous multilayer soft lithography). SBS is a“thermoset elastomer”, meaning that above a certain temperature it meltsand becomes plastic (as opposed to elastic); reducing the temperatureyields the elastomer again. Thus, layers can be bonded together byheating.

[0123] Polyurethanes

[0124] Polyurethanes are produced from di-isocyanates (A-A) anddi-alcohols or di-amines (B-B); since there are a large variety ofdi-isocyanates and di-alcohols/amines, the number of different types ofpolyurethanes is huge. The A vs. B nature of the polymers, however,would make them useful for heterogeneous multilayer soft lithographyjust as RTV 615 is: by using excess A-A in one layer and excess B-B inthe other layer.

[0125] Silicones

[0126] Silicone polymers probably have the greatest structural variety,and almost certainly have the greatest number of commercially availableformulations. The vinyl-to-(Si—H) crosslinking of RTV 615 (which allowsboth heterogeneous multilayer soft lithography and photoresistencapsulation) has already been discussed, but this is only one ofseveral crosslinking methods used in silicone polymer chemistry.

[0127] 5. Operation of Device

[0128]FIGS. 7B and 7H together show the closing of a first flow channelby pressurizing a second flow channel, with FIG. 7B (a front sectionalview cutting through flow channel 32 in corresponding FIG. 7A), showingan open first flow channel 30; with FIG. 7H showing first flow channel30 closed by pressurization of the second flow channel 32.

[0129] Referring to FIG. 7B, first flow channel 30 and second flowchannel 32 are shown. Membrane 25 separates the flow channels, formingthe top of first flow channel 30 and the bottom of second flow channel32. As can be seen, flow channel 30 is “open”.

[0130] As can be seen in FIG. 7H, pressurization of flow channel 32(either by gas or liquid introduced therein) causes membrane 25 todeflect downward, thereby pinching off flow F passing through flowchannel 30. Accordingly, by varying the pressure in channel 32, alinearly actuable valving system is provided such that flow channel 30can be opened or closed by moving membrane 25 as desired. (Forillustration purposes only, channel 30 in FIG. 7G is shown in a “mostlyclosed” position, rather than a “fully closed” position).

[0131] Since such valves are actuated by moving the roof of the channelsthemselves (i.e.: moving membrane 25) valves and pumps produced by thistechnique have a truly zero dead volume, and switching valves made bythis technique have a dead volume approximately equal to the activevolume of the valve, for example about 100×100×10 μm=100 pL. Such deadvolumes and areas consumed by the moving membrane are approximately twoorders of magnitude smaller than known conventional microvalves. Smallerand larger valves and switching valves are contemplated in the presentinvention, and a non-exclusive list of ranges of dead volume includes 1aL to 1 uL, 100 aL to 100 nL, 1 fL to 10 nL, 100 fL to 1 nL, and 1 pL to100 pL.

[0132] The extremely small volumes capable of being delivered by pumpsand valves in accordance with the present invention represent asubstantial advantage. Specifically, the smallest known volumes of fluidcapable of being manually metered is around 0.1 μl. The smallest knownvolumes capable of being metered by automated systems is about ten-timeslarger (1 μl). Utilizing pumps and valves in accordance with the presentinvention, volumes of liquid of 10 nl or smaller can routinely bemetered and dispensed. The accurate metering of extremely small volumesof fluid enabled by the present invention would be extremely valuable ina large number of biological applications, including diagnostic testsand assays.

[0133] Equation 1 represents a highly simplified mathematical model ofdeflection of a rectangular, linear, elastic, isotropic plate of uniformthickness by an applied pressure:

w=(BPb⁴)/(Eh³),  (1)

[0134] where:

[0135] w=deflection of plate;

[0136] B=shape coefficient (dependent upon length vs. width and supportof edges of plate);

[0137] P=applied pressure;

[0138] b=plate width

[0139] E=Young's modulus; and

[0140] h=plate thickness.

[0141] Thus even in this extremely simplified expression, deflection ofan elastomeric membrane in response to a pressure will be a function of:the length, width, and thickness of the membrane, the flexibility of themembrane (Young's modulus), and the applied actuation force. Becauseeach of these parameters will vary widely depending upon the actualdimensions and physical composition of a particular elastomeric devicein accordance with the present invention, a wide range of membranethicknesses and elasticities, channel widths, and actuation forces arecontemplated by the present invention.

[0142] It should be understood that the formula just presented is onlyan approximation, since in general the membrane does not have uniformthickness, the membrane thickness is not necessarily small compared tothe length and width, and the deflection is not necessarily smallcompared to length, width, or thickness of the membrane. Nevertheless,the equation serves as a useful guide for adjusting variable parametersto achieve a desired response of deflection versus applied force.

[0143]FIGS. 8A and 8B illustrate valve opening vs. applied pressure fora 100 μm wide first flow channel 30 and a 50 μm wide second flow channel32. The membrane of this device was formed by a layer of GeneralElectric Silicones RTV 615 having a thickness of approximately 30 μm anda Young's modulus of approximately 750 kPa. FIGS. 21a and 21 b show theextent of opening of the valve to be substantially linear over most ofthe range of applied pressures.

[0144] Air pressure was applied to actuate the membrane of the devicethrough a 10 cm long piece of plastic tubing having an outer diameter of0.025″ connected to a 25 mm piece of stainless steel hypodermic tubingwith an outer diameter of 0.025″ and an inner diameter of 0.013″. Thistubing was placed into contact with the control channel by insertioninto the elastomeric block in a direction normal to the control channel.Air pressure was applied to the hypodermic tubing from an external LHDAminiature solenoid valve manufactured by Lee Co.

[0145] In certain embodiments, it may be useful to apply both positiveand negative pressures to actuate the membrane. For example, where theelastomer material is relatively inflexible, an extremely rapid responsetime is desired, or the flow channel dimensions are small, it may beuseful to apply a positive pressure to a control channel actuate themembrane into the flow channel, followed by a negative pressure to causethe membrane to be displaced out of the flow channel.

[0146] Moreover, it may also be useful to cause movement of fluidthrough the microfluidic device by the direct application of pressure tothe flow channel, such as the application of positive pressure directlyto the flow channel inlet, or application of negative pressure directlyto the flow channel outlet. Direct application of pressure alone candrive the flow of fluid within the microfluidic device, or directpressure may be utilized in conjunction with actuation of membranesoverlying the flow channel. Pressure applied directly to the flowchannel can also serve to alter the speed of movement of materialsthought the flow channel as desired.

[0147] While control of the flow of material through the device has sofar been described utilizing applied gas pressure, other fluids could beused. For example, air is compressible, and thus experiences some finitedelay between the time of application of pressure by the externalsolenoid valve and the time that this pressure is experienced by themembrane. In an alternative embodiment of the present invention,pressure could be applied from an external source to a noncompressiblefluid such as water or hydraulic oils, resulting in a near-instantaneoustransfer of applied pressure to the membrane. However, if the displacedvolume of the valve is large or the control channel is narrow, higherviscosity of a control fluid may contribute to delay in actuation. Theoptimal medium for transferring pressure will therefore depend upon theparticular application and device configuration, and both gaseous andliquid media are contemplated by the invention.

[0148] While external applied pressure as described above has beenapplied by a pump/tank system through a pressure regulator and externalminiature valve, other methods of applying external pressure are alsocontemplated in the present invention, including gas tanks, compressors,piston systems, and columns of liquid. Also contemplated is the use ofnaturally occurring pressure sources such as may be found inside livingorganisms, such as blood pressure, gastric pressure, the pressurepresent in the cerebro-spinal fluid, pressure present in theintra-ocular space, and the pressure exerted by muscles during normalflexure. Other methods of regulating external pressure are alsocontemplated, such as miniature valves, pumps, macroscopic peristalticpumps, pinch valves, and other types of fluid regulating equipment suchas is known in the art.

[0149] As can be seen, the response of valves in accordance withembodiments of the present invention have been experimentally shown tobe almost perfectly linear over a large portion of its range of travel,with minimal hysteresis. Accordingly, the present valves are ideallysuited for microfluidic metering and fluid control. The linearity of thevalve response demonstrates that the individual valves are well modeledas Hooke's Law springs. Furthermore, high pressures in the flow channel(i.e.: back pressure) can be countered simply by increasing theactuation pressure. Experimentally, the present inventors have achievedvalve closure at back pressures of 70 kPa, but higher pressures are alsocontemplated. The following is a nonexclusive list of pressure rangesencompassed by the present invention: 10 Pa-25 MPa; 100 Pa-10 Mpa, 1kPa-1 MPa, 1 kPa-300 kPa, 5 kPa-200 kPa, and 15 kPa-100 kPa.

[0150] While valves and pumps do not require linear actuation to openand close, linear response does allow valves to more easily be used asmetering devices. In one embodiment of the invention, the opening of thevalve is used to control flow rate by being partially actuated to aknown degree of closure. Linear valve actuation makes it easier todetermine the amount of actuation force required to close the valve to adesired degree of closure. Another benefit of linear actuation is thatthe force required for valve actuation may be easily determined from thepressure in the flow channel. If actuation is linear, increased pressurein the flow channel may be countered by adding the same pressure (forceper unit area) to the actuated portion of the valve.

[0151] Linearity of a valve depends on the structure, composition, andmethod of actuation of the valve structure. Furthermore, whetherlinearity is a desirable characteristic in a valve depends on theapplication. Therefore, both linearly and non-linearly actuable valvesare contemplated in the present invention, and the pressure ranges overwhich a valve is linearly actuable will vary with the specificembodiment.

[0152]FIG. 9 illustrates time response (i.e.: closure of valve as afunction of time in response to a change in applied pressure) of a 100μm×100 μm×10 μm RTV microvalve with 10-cm-long air tubing connected fromthe chip to a pneumatic valve as described above.

[0153] Two periods of digital control signal, actual air pressure at theend of the tubing and valve opening are shown in FIG. 9. The pressureapplied on the control line is 100 kPa, which is substantially higherthan the ˜40 kPa required to close the valve. Thus, when closing, thevalve is pushed closed with a pressure 60 kPa greater than required.When opening, however, the valve is driven back to its rest positiononly by its own spring force (≦40 kPa). Thus, τ_(close) is expected tobe smaller than τ_(open). There is also a lag between the control signaland control pressure response, due to the limitations of the miniaturevalve used to control the pressure. Calling such lags t and the 1/e timeconstants τ, the values are: t_(open)=3.63 ms, τ_(open)=1.88 ms,t_(close)=2.15 ms, τ_(close)=0.51 ms. If 3τ each are allowed for openingand closing, the valve runs comfortably at 75 Hz when filled withaqueous solution.

[0154] If one used another actuation method which did not suffer fromopening and closing lag, this valve would run at ˜375 Hz. Note also thatthe spring constant can be adjusted by changing the membrane thickness;this allows optimization for either fast opening or fast closing. Thespring constant could also be adjusted by changing the elasticity(Young's modulus) of the membrane, as is possible by introducing dopantinto the membrane or by utilizing a different elastomeric material toserve as the membrane (described above in conjunction with FIGS. 7C-7H.)

[0155] When experimentally measuring the valve properties as illustratedin FIG. 9 the valve opening was measured by fluorescence. In theseexperiments, the flow channel was filled with a solution of fluoresceinisothiocyanate (FITC) in buffer (pH≧8) and the fluorescence of a squarearea occupying the center ˜⅓rd of the channel is monitored on anepi-fluorescence microscope with a photomultiplier tube with a 10 kHzbandwidth. The pressure was monitored with a Wheatstone-bridge pressuresensor (SenSym SCC15GD2) pressurized simultaneously with the controlline through nearly identical pneumatic connections.

[0156] 6. Flow Channel Cross Sections

[0157] The flow channels of the present invention may optionally bedesigned with different cross sectional sizes and shapes, offeringdifferent advantages, depending upon their desired application. Forexample, the cross sectional shape of the lower flow channel may have acurved upper surface, either along its entire length or in the regiondisposed under an upper cross channel). Such a curved upper surfacefacilitates valve sealing, as follows.

[0158] Referring to FIG. 10, a cross sectional view (similar to that ofFIG. 7B) through flow channels 30 and 32 is shown. As can be seen, flowchannel 30 is rectangular in cross sectional shape. In an alternatepreferred aspect of the invention, as shown in FIG. 20, thecross-section of a flow channel 30 instead has an upper curved surface.

[0159] Referring first to FIG. 10, when flow channel 32 is pressurized,the membrane portion 25 of elastomeric block 24 separating flow channels30 and 32 will move downwardly to the successive positions shown by thedotted lines 25A, 25B, 25C, 25D, and 25E. As can be seen, incompletesealing may possibly result at the edges of flow channel 30 adjacentplanar substrate 14.

[0160] In the alternate preferred embodiment of FIG. 11, flow channel 30a has a curved upper wall 25A. When flow channel 32 is pressurized,membrane portion 25 will move downwardly to the successive positionsshown by dotted lines 25A2, 25A3, 25A4 and 25A5, with edge portions ofthe membrane moving first into the flow channel, followed by topmembrane portions. An advantage of having such a curved upper surface atmembrane 25A is that a more complete seal will be provided when flowchannel 32 is pressurized. Specifically, the upper wall of the flowchannel 30 will provide a continuous contacting edge against planarsubstrate 14, thereby avoiding the “island” of contact seen between wall25 and the bottom of flow channel 30 in FIG. 10.

[0161] Another advantage of having a curved upper flow channel surfaceat membrane 25A is that the membrane can more readily conform to theshape and volume of the flow channel in response to actuation.Specifically, where a rectangular flow channel is employed, the entireperimeter (2×flow channel height, plus the flow channel width) must beforced into the flow channel. However where an arched flow channel isused, a smaller perimeter of material (only the semi-circular archedportion) must be forced into the channel. In this manner, the membranerequires less change in perimeter for actuation and is therefore moreresponsive to an applied actuation force to block the flow channel.

[0162] In an alternate aspect, (not illustrated), the bottom of flowchannel 30 is rounded such that its curved surface mates with the curvedupper wall 25A as seen in FIG. 20 described above.

[0163] In summary, the actual conformational change experienced by themembrane upon actuation will depend upon the configuration of theparticular elastomeric structure. Specifically, the conformationalchange will depend upon the length, width, and thickness profile of themembrane, its attachment to the remainder of the structure, and theheight, width, and shape of the flow and control channels and thematerial properties of the elastomer used. The conformational change mayalso depend upon the method of actuation, as actuation of the membranein response to an applied pressure will vary somewhat from actuation inresponse to a magnetic or electrostatic force.

[0164] Moreover, the desired conformational change in the membrane willalso vary depending upon the particular application for the elastomericstructure. In the simplest embodiments described above, the valve mayeither be open or closed, with metering to control the degree of closureof the valve. In other embodiments however, it may be desirable to alterthe shape of the membrane and/or the flow channel in order to achievemore complex flow regulation. For instance, the flow channel could beprovided with raised protrusions beneath the membrane portion, such thatupon actuation the membrane shuts off only a percentage of the flowthrough the flow channel, with the percentage of flow blockedinsensitive to the applied actuation force.

[0165] Many membrane thickness profiles and flow channel cross-sectionsare contemplated by the present invention, including rectangular,trapezoidal, circular, ellipsoidal, parabolic, hyperbolic, andpolygonal, as well as sections of the above shapes. More complexcross-sectional shapes, such as the embodiment with protrusionsdiscussed immediately above or an embodiment having concavities in theflow channel, are also contemplated by the present invention.

[0166] In addition, while the invention is described primarily above inconjunction with an embodiment wherein the walls and ceiling of the flowchannel are formed from elastomer, and the floor of the channel isformed from an underlying substrate, the present invention is notlimited to this particular orientation. Walls and floors of channelscould also be formed in the underlying substrate, with only the ceilingof the flow channel constructed from elastomer. This elastomer flowchannel ceiling would project downward into the channel in response toan applied actuation force, thereby controlling the flow of materialthrough the flow channel. In general, monolithic elastomer structures asdescribed elsewhere in the instant application are preferred formicrofluidic applications. However, it may be useful to employ channelsformed in the substrate where such an arrangement provides advantages.For instance, a substrate including optical waveguides could beconstructed so that the optical waveguides direct light specifically tothe side of a microfluidic channel.

[0167] 7. Alternate Valve Actuation Techniques

[0168] In addition to pressure based actuation systems described above,optional electrostatic and magnetic actuation systems are alsocontemplated, as follows.

[0169] Electrostatic actuation can be accomplished by forming oppositelycharged electrodes (which will tend to attract one another when avoltage differential is applied to them) directly into the monolithicelastomeric structure. For example, referring to FIG. 7B, an optionalfirst electrode 70 (shown in phantom) can be positioned on (or in)membrane 25 and an optional second electrode 72 (also shown in phantom)can be positioned on (or in) planar substrate 14. When electrodes 70 and72 are charged with opposite polarities, an attractive force between thetwo electrodes will cause membrane 25 to deflect downwardly, therebyclosing the “valve” (i.e.: closing flow channel 30).

[0170] For the membrane electrode to be sufficiently conductive tosupport electrostatic actuation, but not so mechanically stiff so as toimpede the valve's motion, a sufficiently flexible electrode must beprovided in or over membrane 25. Such an electrode may be provided by athin metallization layer, doping the polymer with conductive material,or making the surface layer out of a conductive material.

[0171] In an exemplary aspect, the electrode present at the deflectingmembrane can be provided by a thin metallization layer which can beprovided, for example, by sputtering a thin layer of metal such as 20 nmof gold. In addition to the formation of a metallized membrane bysputtering, other metallization approaches such as chemical epitaxy,evaporation, electroplating, and electroless plating are also available.Physical transfer of a metal layer to the surface of the elastomer isalso available, for example by evaporating a metal onto a flat substrateto which it adheres poorly, and then placing the elastomer onto themetal and peeling the metal off of the substrate.

[0172] A conductive electrode 70 may also be formed by depositing carbonblack (i.e. Cabot Vulcan XC72R) on the elastomer surface, either bywiping on the dry powder or by exposing the elastomer to a suspension ofcarbon black in a solvent which causes swelling of the elastomer, (suchas a chlorinated solvent in the case of PDMS). Alternatively, theelectrode 70 may be formed by constructing the entire layer 20 out ofelastomer doped with conductive material (i.e. carbon black or finelydivided metal particles). Yet further alternatively, the electrode maybe formed by electrostatic deposition, or by a chemical reaction thatproduces carbon. In experiments conducted by the present inventors,conductivity was shown to increase with carbon black concentration from5.6×10⁻¹⁶ to about 5×10⁻³ (Ω-cm)⁻¹. The lower electrode 72, which is notrequired to move, may be either a compliant electrode as describedabove, or a conventional electrode such as evaporated gold, a metalplate, or a doped semiconductor electrode.

[0173] Magnetic actuation of the flow channels can be achieved byfabricating the membrane separating the flow channels with amagnetically polarizable material such as iron, or a permanentlymagnetized material such as polarized NdFeB. In experiments conducted bythe present inventors, magnetic silicone was created by the addition ofiron powder (about 1 μm particle size), up to 20% iron by weight.

[0174] Where the membrane is fabricated with a magnetically polarizablematerial, the membrane can be actuated by attraction in response to anapplied magnetic field Where the membrane is fabricated with a materialcapable of maintaining permanent magnetization, the material can firstbe magnetized by exposure to a sufficiently high magnetic field, andthen actuated either by attraction or repulsion in response to thepolarity of an applied inhomogenous magnetic field.

[0175] The magnetic field causing actuation of the membrane can begenerated in a variety of ways. In one embodiment, the magnetic field isgenerated by an extremely small inductive coil formed in or proximate tothe elastomer membrane. The actuation effect of such a magnetic coilwould be localized, allowing actuation of individual pump and/or valvestructures. Alternatively, the magnetic field could be generated by alarger, more powerful source, in which case actuation would be globaland would actuate multiple pump and/or valve structures at one time.

[0176] It is also possible to actuate the device by causing a fluid flowin the control channel based upon the application of thermal energy,either by thermal expansion or by production of gas from liquid. Forexample, in one alternative embodiment in accordance with the presentinvention, a pocket of fluid (e.g. in a fluid-filled control channel) ispositioned over the flow channel. Fluid in the pocket can be incommunication with a temperature variation system, for example a heater.Thermal expansion of the fluid, or conversion of material from theliquid to the gas phase, could result in an increase in pressure,closing the adjacent flow channel. Subsequent cooling of the fluid wouldrelieve pressure and permit the flow channel to open.

[0177] 8. Networked Systems

[0178]FIGS. 12A and 12B show a views of a single on/off valve, identicalto the systems set forth above, (for example in FIG. 7A). FIGS. 13A and13B shows a peristaltic pumping system comprised of a plurality of thesingle addressable on/off valves as seen in FIG. 12, but networkedtogether. FIG. 14 is a graph showing experimentally achieved pumpingrates vs. frequency for the peristaltic pumping system of FIG. 13. FIGS.15A and 15B show a schematic view of a plurality of flow channels whichare controllable by a single control line. This system is also comprisedof a plurality of the single addressable on/off valves of FIG. 12,multiplexed together, but in a different arrangement than that of FIG.12. FIG. 16 is a schematic illustration of a multiplexing system adaptedto permit fluid flow through selected channels, comprised of a pluralityof the single on/off valves of FIG. 12, joined or networked together.

[0179] Referring first to FIGS. 12A and 12B, a schematic of flowchannels 30 and 32 is shown. Flow channel 30 preferably has a fluid (orgas) flow F passing therethrough. Flow channel 32, (which crosses overflow channel 30, as was already explained herein), is pressurized suchthat membrane 25 separating the flow channels may be depressed into thepath of flow channel 30, shutting off the passage of flow Ftherethrough, as has been explained. As such, “flow channel” 32 can alsobe referred to as a “control line” which actuates a single valve in flowchannel 30. In FIGS. 12 to 15, a plurality of such addressable valvesare joined or networked together in various arrangements to producepumps, capable of peristaltic pumping, and other fluidic logicapplications.

[0180] Referring to FIG. 13A and 13B, a system for peristaltic pumpingis provided, as follows. A flow channel 30 has a plurality of generallyparallel flow channels (i.e.: control lines) 32A, 32B and 32C passingthereover. By pressurizing control line 32A, flow F through flow channel30 is shut off under membrane 25A at the intersection of control line32A and flow channel 30. Similarly, (but not shown), by pressurizingcontrol line 32B, flow F through flow channel 30 is shut off undermembrane 25B at the intersection of control line 32B and flow channel30, etc.

[0181] Each of control lines 32A, 32B, and 32C is separatelyaddressable. Therefore, peristalsis may be actuated by the pattern ofactuating 32A and 32C together, followed by 32A, followed by 32A and 32Btogether, followed by 32B, followed by 32B and C together, etc. Thiscorresponds to a successive “101, 100, 110, 010, 011, 001” pattern,where “0” indicates “valve open” and “1” indicates “valve closed.” Thisperistaltic pattern is also known as a 120° pattern (referring to thephase angle of actuation between three valves). Other peristalticpatterns are equally possible, including 60° and 90° patterns.

[0182] In experiments performed by the inventors, a pumping rate of 2.35nL/s was measured by measuring the distance traveled by a column ofwater in thin (0.5 mm i.d.) tubing; with 100×100×10 μm valves under anactuation pressure of 40 kPa. The pumping rate increased with actuationfrequency until approximately 75 Hz, and then was nearly constant untilabove 200 Hz. The valves and pumps are also quite durable and theelastomer membrane, control channels, or bond have never been observedto fail. In experiments performed by the inventors, none of the valvesin the peristaltic pump described herein show any sign of wear orfatigue after more than 4 million actuations. In addition to theirdurability, they are also gentle. A solution of E. Coli pumped through achannel and tested for viability showed a 94% survival rate.

[0183]FIG. 14 is a graph showing experimentally achieved pumping ratesvs. frequency for the peristaltic pumping system of FIG. 13.

[0184]FIGS. 15A and 15B illustrates another way of assembling aplurality of the addressable valves of FIG. 12. Specifically, aplurality of parallel flow channels 30A, 30B, and 30C are provided. Flowchannel (i.e.: control line) 32 passes thereover across flow channels30A, 30B, and 30C. Pressurization of control line 32 simultaneouslyshuts off flows F1, F2 and F3 by depressing membranes 25A, 25B, and 25Clocated at the intersections of control line 32 and flow channels 30A,30B, and 30C.

[0185]FIG. 16 is a schematic illustration of a multiplexing systemadapted to selectively permit fluid to flow through selected channels,as follows. The downward deflection of membranes separating therespective flow channels from a control line passing thereabove (forexample, membranes 25A, 25B, and 25C in FIGS. 15A and 15B) dependsstrongly upon the membrane dimensions. Accordingly, by varying thewidths of flow channel control line 32 in FIGS. 15A and 15B, it ispossible to have a control line pass over multiple flow channels, yetonly actuate (i.e.: seal) desired flow channels. FIG. 16 illustrates aschematic of such a system, as follows.

[0186] A plurality of parallel flow channels 30A, 30B, 30C, 30D, 30E and30F are positioned under a plurality of parallel control lines 32A, 32B,32C, 32D, 32E and 32F. Control channels 32A, 32B, 32C, 32D, 32E and 32Fare adapted to shut off fluid flows F1, F2, F3, F4, F5 and F6 passingthrough parallel flow channels 30A, 30B, 30C, 30D, 30E and 30F using anyof the valving systems described above, with the following modification.

[0187] Each of control lines 32A, 32B, 32C, 32D, 32E and 32F have bothwide and narrow portions. For example, control line 32A is wide inlocations disposed over flow channels 30A, 30C and 30E. Similarly,control line 32B is wide in locations disposed over flow channels 30B,30D and 30F, and control line 32C is wide in locations disposed overflow channels 30A, 30B, 30E and 30F.

[0188] At the locations where the respective control line is wide, itspressurization will cause the membrane (25) separating the flow channeland the control line to depress significantly into the flow channel,thereby blocking the flow passage therethrough. Conversely, in thelocations where the respective control line is narrow, membrane (25)will also be narrow. Accordingly, the same degree of pressurization willnot result in membrane (25) becoming depressed into the flow channel(30). Therefore, fluid passage thereunder will not be blocked.

[0189] For example, when control line 32A is pressurized, it will blockflows F1, F3 and F5 in flow channels 30A, 30C and 30E. Similarly, whencontrol line 32C is pressurized, it will block flows F1, F2, F5 and F6in flow channels 30A, 30B, 30E and 30F. As can be appreciated, more thanone control line can be actuated at the same time. For example, controllines 32A and 32C can be pressurized simultaneously to block all fluidflow except F4 (with 32A blocking F1, F3 and F5; and 32C blocking F1,F2, F5 and F6).

[0190] By selectively pressurizing different control lines (32) bothtogether and in various sequences, a great degree of fluid flow controlcan be achieved. Moreover, by extending the present system to more thansix parallel flow channels (30) and more than four parallel controllines (32), and by varying the positioning of the wide and narrowregions of the control lines, very complex fluid flow control systemsmay be fabricated. A property of such systems is that it is possible toturn on any one flow channel out of n flow channels with only 2(log₂n)control lines.

[0191] 9. Selectively Addressable Reaction Chambers Along Flow Lines

[0192] In a further embodiment of the invention, illustrated in FIGS.17A, 17B, 17C and 17D, a system for selectively directing fluid flowinto one more of a plurality of reaction chambers disposed along a flowline is provided.

[0193]FIG. 17A shows a top view of a flow channel 30 having a pluralityof reaction chambers 80A and 80B disposed therealong. Preferably flowchannel 30 and reaction chambers 80A and 80B are formed together asrecesses into the bottom surface of a first layer 100 of elastomer.

[0194]FIG. 17B shows a bottom plan view of another elastomeric layer 110with two control lines 32A and 32B each being generally narrow, buthaving wide extending portions 33A and 33B formed as recesses therein.

[0195] As seen in the exploded view of FIG. 17C, and assembled view ofFIG. 17D, elastomeric layer 110 is placed over elastomeric layer 100.Layers 100 and 110 are then bonded together, and the integrated systemoperates to selectively direct fluid flow F (through flow channel 30)into either or both of reaction chambers 80A and 80B, as follows.Pressurization of control line 32A will cause the membrane 25 (i.e.: thethin portion of elastomer layer 100 located below extending portion 33Aand over regions 82A of reaction chamber 80A) to become depressed,thereby shutting off fluid flow passage in regions 82A, effectivelysealing reaction chamber 80 from flow channel 30. As can also be seen,extending portion 33A is wider than the remainder of control line 32A.As such, pressurization of control line 32A will not result in controlline 32A sealing flow channel 30.

[0196] As can be appreciated, either or both of control lines 32A and32B can be actuated at once. When both control lines 32A and 32B arepressurized together, sample flow in flow channel 30 will enter neitherof reaction chambers 80A or 80B.

[0197] The concept of selectably controlling fluid introduction intovarious addressable reaction chambers disposed along a flow line (FIGS.17A-D) can be combined with concept of selectably controlling fluid flowthrough one or more of a plurality of parallel flow lines (FIG. 16) toyield a system in which a fluid sample or samples can be can be sent toany particular reaction chamber in an array of reaction chambers. Anexample of such a system is provided in FIG. 18, in which parallelcontrol channels 32A, 32B and 32C with extending portions 34 (all shownin phantom) selectively direct fluid flows F1 and F2 into any of thearray of reaction wells 80A, 80B, 80C or 80D as explained above; whilepressurization of control lines 32C and 32D selectively shuts off flowsF2 and F1, respectively.

[0198] In yet another novel embodiment, fluid passage between parallelflow channels is possible. Referring to FIG. 19, either or both ofcontrol lines 32A or 32D can be depressurized such that fluid flowthrough lateral passageways 35 (between parallel flow channels 30A and30B) is permitted. In this aspect of the invention, pressurization ofcontrol lines 32C and 32D would shut flow channel 30A between 35A and35B, and would also shut lateral passageways 35B. As such, flow enteringas flow F1 would sequentially travel through 30A, 35A and leave 30B asflow F4.

[0199] 10. Switchable Flow Arrays

[0200] In yet another novel embodiment, fluid passage can be selectivelydirected to flow in either of two perpendicular directions. An exampleof such a “switchable flow array” system is provided in FIGS. 20A to20D. FIG. 20A shows a bottom view of a first layer of elastomer 90, (orany other suitable substrate), having a bottom surface with a pattern ofrecesses forming a flow channel grid defined by an array of solid posts92, each having flow channels passing therearound.

[0201] In preferred aspects, an additional layer of elastomer is boundto the top surface of layer 90 such that fluid flow can be selectivelydirected to move either in direction F1, or perpendicular direction F2.FIG. 20 is a bottom view of the bottom surface of the second layer ofelastomer 95 showing recesses formed in the shape of alternating“vertical” control lines 96 and “horizontal” control lines 94.“Vertical” control lines 96 have the same width therealong, whereas“horizontal” control lines 94 have alternating wide and narrow portions,as shown.

[0202] Elastomeric layer 95 is positioned over top of elastomeric layer90 such that “vertical” control lines 96 are positioned over posts 92 asshown in FIG. 20C and “horizontal” control lines 94 are positioned withtheir wide portions between posts 92, as shown in FIG. 20D.

[0203] As can be seen in FIG. 20C, when “vertical” control lines 96 arepressurized, the membrane of the integrated structure formed by theelastomeric layer initially positioned between layers 90 and 95 inregions 98 will be deflected downwardly over the array of flow channelssuch that flow in only able to pass in flow direction F2 (i.e.:vertically), as shown.

[0204] As can be seen in FIG. 20D, when “horizontal” control lines 94are pressurized, the membrane of the integrated structure formed by theelastomeric layer initially positioned between layers 90 and 95 inregions 99 will be deflected downwardly over the array of flow channels,(but only in the regions where they are widest), such that flow in onlyable to pass in flow direction F1 (i.e.: horizontally), as shown.

[0205] The design illustrated in FIGS. 20 allows a switchable flow arrayto be constructed from only two elastomeric layers, with no verticalvias passing between control lines in different elastomeric layersrequired. If all vertical flow control lines 94 are connected, they maybe pressurized from one input. The same is true for all horizontal flowcontrol lines 96.

[0206] 11. Normally-Closed Valve Structure

[0207]FIGS. 7B and 7H above depict a valve structure in which theelastomeric membrane is moveable from a first relaxed position to asecond actuated position in which the flow channel is blocked. However,the present invention is not limited to this particular valveconfiguration.

[0208] FIGS. 21A-21J show a variety of views of a normally-closed valvestructure in which the elastomeric membrane is moveable from a firstrelaxed position blocking a flow channel, to a second actuated positionin which the flow channel is open, utilizing a negative controlpressure.

[0209]FIG. 21A shows a plan view, and FIG. 21B shows a cross sectionalview along line 42B-42B′, of normally-closed valve 4200 in an unactuatedstate. Flow channel 4202 and control channel 4204 are formed inelastomeric block 4206 overlying substrate 4205. Flow channel 4202includes a first portion 4202 a and a second portion 4202 b separated byseparating portion 4208. Control channel 4204 overlies separatingportion 4208. As shown in FIG. 42B, in its relaxed, unactuated position,separating portion 4008 remains positioned between flow channel portions4202 a and 4202 b, interrupting flow channel 4202.

[0210]FIG. 21C shows a cross-sectional view of valve 4200 whereinseparating portion 4208 is in an actuated position. When the pressurewithin control channel 4204 is reduced to below the pressure in the flowchannel (for example by vacuum pump), separating portion 4208experiences an actuating force drawing it into control channel 4204. Asa result of this actuation force membrane 4208 projects into controlchannel 4204, thereby removing the obstacle to a flow of materialthrough flow channel 4202 and creating a passageway 4203. Upon elevationof pressure within control channel 4204, separating portion 4208 willassume its natural position, relaxing back into and obstructing flowchannel 4202.

[0211] The behavior of the membrane in response to an actuation forcemay be changed by varying the width of the overlying control channel.Accordingly, FIGS. 21D-42H show plan and cross-sectional views of analternative embodiment of a normally-closed valve 4201 in which controlchannel 4207 is substantially wider than separating portion 4208. Asshown in cross-sectional views FIG. 21E-F along line 42E-42E′ of FIG.21D, because a larger area of elastomeric material is required to bemoved during actuation, the actuation force necessary to be applied isreduced.

[0212]FIGS. 21G and H show a cross-sectional views along line 40G-40G′of FIG. 21D. In comparison with the unactuated valve configuration shownin FIG. 21G, FIG. 21H shows that reduced pressure within wider controlchannel 4207 may under certain circumstances have the unwanted effect ofpulling underlying elastomer 4206 away from substrate 4205, therebycreating undesirable void 4212.

[0213] Accordingly, FIG. 211 shows a plan view, and FIG. 21J shows across-sectional view along line 21J-21J′ of FIG. 21I, of valve structure4220 which avoids this problem by featuring control line 4204 with aminimum width except in segment 4204 a overlapping separating portion4208. As shown in FIG. 21J, even under actuated conditions the narrowercross-section of control channel 4204 reduces the attractive force onthe underlying elastomer material 4206, thereby preventing thiselastomer material from being drawn away from substrate 4205 andcreating an undesirable void.

[0214] While a normally-closed valve structure actuated in response topressure is shown in FIGS. 21A-21J, a normally-closed valve inaccordance with the present invention is not limited to thisconfiguration. For example, the separating portion obstructing the flowchannel could alternatively be manipulated by electric or magneticfields, as described extensively above.

[0215] 12. Side-Actuated Valve

[0216] While the above description has focused upon microfabricatedelastomeric valve structures in which a control channel is positionedabove and separated by an intervening elastomeric membrane from anunderlying flow channel, the present invention is not limited to thisconfiguration. FIGS. 22A and 22B show plan views of one embodiment of aside-actuated valve structure in accordance with one embodiment of thepresent invention.

[0217]FIG. 22A shows side-actuated valve structure 4800 in an unactuatedposition. Flow channel 4802 is formed in elastomeric layer 4804. Controlchannel 4806 abutting flow channel 4802 is also formed in elastomericlayer 4804. Control channel 4806 is separated from flow channel 4802 byelastomeric membrane portion 4808. A second elastomeric layer (notshown) is bonded over bottom elastomeric layer 4804 to enclose flowchannel 4802 and control channel 4806.

[0218]FIG. 22B shows side-actuated valve structure 4800 in an actuatedposition. In response to a build up of pressure within control channel4806, membrane 4808 deforms into flow channel 4802, blocking flowchannel 4802. Upon release of pressure within control channel 4806,membrane 4808 would relax back into control channel 4806 and open flowchannel 4802.

[0219] While a side-actuated valve structure actuated in response topressure is shown in FIGS. 22A and 22B, a side-actuated valve inaccordance with the present invention is not limited to thisconfiguration. For example, the elastomeric membrane portion locatedbetween the abutting flow and control channels could alternatively bemanipulated by electric or magnetic fields, as described extensivelyabove.

[0220] 13. Composite Structures

[0221] Microfabricated elastomeric structures of the present inventionmay be combined with non-elastomeric materials to create compositestructures. FIG. 23 shows a cross-sectional view of one embodiment of acomposite structure in accordance with the present invention. FIG. 23shows composite valve structure 5700 including first, thin elastomerlayer 5702 overlying semiconductor-type substrate 5704 having channel5706 formed therein. Second, thicker elastomer layer 5708 overlies firstelastomer layer 5702. Actuation of first elastomer layer 5702 to driveit into channel 5706, will cause composite structure 5700 to operate asa valve.

[0222]FIG. 24 shows a cross-sectional view of a variation on this theme,wherein thin elastomer layer 5802 is sandwiched between two hard,semiconductor substrates 5804 and 5806, with lower substrate 5804featuring channel 5808. Again, actuation of thin elastomer layer 5802 todrive it into channel 5808 will cause composite structure 5810 tooperate as a valve.

[0223] The structures shown in FIGS. 23 or 24 may be fabricatedutilizing either the multilayer soft lithography or encapsulationtechniques described above. In the multilayer soft lithography method,the elastomer layer(s) would be formed and then placed over thesemiconductor substrate bearing the channel. In the encapsulationmethod, the channel would be first formed in the semiconductorsubstrate, and then the channel would be filled with a sacrificialmaterial such as photoresist. The elastomer would then be formed inplace over the substrate, with removal of the sacrificial materialproducing the channel overlaid by the elastomer membrane. As isdiscussed in detail below in connection with bonding of elastomer toother types of materials, the encapsulation approach may result in astronger seal between the elastomer membrane component and theunderlying nonelastomer substrate component.

[0224] As shown in FIGS. 23 and 24, a composite structure in accordancewith embodiments of the present invention may include a hard substratethat bears a passive feature such as a channels. However, the presentinvention is not limited to this approach, and the underlying hardsubstrate may bear active features that interact with an elastomercomponent bearing a recess. This is shown in FIG. 25, wherein compositestructure 5900 includes elastomer component 5902 containing recess 5904having walls 5906 and ceiling 5908. Ceiling 5908 forms flexible membraneportion 5909. Elastomer component 5902 is sealed against substantiallyplanar nonelastomeric component 5910 that includes active device 5912.Active device 5912 may interact with material present in recess 5904and/or flexible membrane portion 5909.

[0225] Many types of active structures may be present in thenonelastomer substrate. Active structures that could be present in anunderlying hard substrate include, but are not limited to, resistors,capacitors, photodiodes, transistors, chemical field effect transistors(chem FET's), amperometric/coulometric electrochemical sensors, fiberoptics, fiber optic interconnects, light emitting diodes, laser diodes,vertical cavity surface emitting lasers (VCSEL's), micromirrors,accelerometers, pressure sensors, flow sensors, CMOS imaging arrays, CCDcameras, electronic logic, microprocessors, thermistors, Peltiercoolers, waveguides, resistive heaters, chemical sensors, strain gauges,inductors, actuators (including electrostatic, magnetic,electromagnetic, bimetallic, piezoelectric, shape-memory-alloy based,and others), coils, magnets, electromagnets, magnetic sensors (such asthose used in hard drives, superconducting quantum interference devices(SQUIDS) and other types), radio frequency sources and receivers,microwave frequency sources and receivers, sources and receivers forother regions of the electromagnetic spectrum, radioactive particlecounters, and electrometers.

[0226] As is well known in the art, a vast variety of technologies canbe utilized to fabricate active features in semiconductor and othertypes of hard substrates, including but not limited printed circuitboard (PCB) technology, CMOS, surface micromachining, bulkmicromachining, printable polymer electronics, and TFT and otheramorphous/polycrystalline techniques as are employed to fabricate laptopand flat screen displays.

[0227] A variety of approaches can be employed to seal the elastomericstructure against the nonelastomeric substrate, ranging from thecreation of a Van der Waals bond between the elastomeric andnonelastomeric components, to creation of covalent or ionic bondsbetween the elastomeric and nonelastomeric components of the compositestructure. Example approaches to sealing the components together arediscussed below, approximately in order of increasing strength.

[0228] A first approach is to rely upon the simple hermetic sealresulting from Van der Waals bonds formed when a substantially planarelastomer layer is placed into contact with a substantially planar layerof a harder, non-elastomer material. In one embodiment, bonding of RTVelastomer to a glass substrate created a composite structure capable ofwithstanding up to about 3-4 psi of pressure. This may be sufficient formany potential applications.

[0229] A second approach is to utilize a liquid layer to assist inbonding. One example of this involves bonding elastomer to a hard glasssubstrate, wherein a weakly acidic solution (5 μl HCl in H₂O, pH 2) wasapplied to a glass substrate. The elastomer component was then placedinto contact with the glass substrate, and the composite structure bakedat 37° C. to remove the water. This resulted in a bond between elastomerand non-elastomer able to withstand a pressure of about 20 psi. In thiscase, the acid may neutralize silanol groups present on the glasssurface, permitting the elastomer and non-elastomer to enter into goodVan der Waals contact with each other.

[0230] Exposure to ethanol can also cause device components to adheretogether. In one embodiment, an RTV elastomer material and a glasssubstrate were washed with ethanol and then dried under Nitrogen. TheRTV elastomer was then placed into contact with the glass and thecombination baked for 3 hours at 80° C. Optionally, the RTV may also beexposed to a vacuum to remove any air bubbles trapped between the slideand the RTV. The strength of the adhesion between elastomer and glassusing this method has withstood pressures in excess of 35 psi. Theadhesion created using this method is not permanent, and the elastomermay be peeled off of the glass, washed, and resealed against the glass.This ethanol washing approach can also be employed used to causesuccessive layers of elastomer to bond together with sufficient strengthto resist a pressure of 30 psi. In alternative embodiments, chemicalssuch as other alcohols or diols could be used to promote adhesionbetween layers.

[0231] An embodiment of a method of promoting adhesion between layers ofa microfabricated structure in accordance with the present inventioncomprises exposing a surface of a first component layer to a chemical,exposing a surface of a second component layer to the chemical, andplacing the surface of the first component layer into contact with thesurface of the second elastomer layer.

[0232] A third approach is to create a covalent chemical bond betweenthe elastomer component and functional groups introduced onto thesurface of a nonelastomer component. Examples of derivitization of anonelastomer substrate surface to produce such functional groups includeexposing a glass substrate to agents such as vinyl silane oraminopropyltriethoxy silane (APTES), which may be useful to allowbonding of the glass to silicone elastomer and polyurethane elastomermaterials, respectively.

[0233] A fourth approach is to create a covalent chemical bond betweenthe elastomer component and a functional group native to the surface ofthe nonelastomer component. For example, RTV elastomer can be createdwith an excess of vinyl groups on its surface. These vinyl groups can becaused to react with corresponding functional groups present on theexterior of a hard substrate material, for example the Si—H bondsprevalent on the surface of a single crystal silicon substrate afterremoval of native oxide by etching. In this example, the strength of thebond created between the elastomer component and the nonelastomercomponent has been observed to exceed the materials strength of theelastomer components.

[0234] II. Damper Structure

[0235] Embodiments of apparatuses and methods in accordance with thepresent invention are directed to microfluidic devices comprising pumps,valves, and fluid oscillation dampers. In this respect, the microfluidicdevices of the present invention is similar to that described in Ungeret al. Science, 2000, 288, 113-116, which is incorporated herein byreference in its entirety. However, microfluidic devices of the presentinvention may further comprise a damper.

[0236] The advantages of microfluidic devices of the present inventioninclude reduced fluid oscillation within a flow channel which reducespotential variability in detection means. As the fluid is pushed throughthe flow channel by the pumps, there is a tendency for the fluid tooscillate, i.e., the fluid is pushed through the flow channel in asinusoidal wave-like fashion. As this oscillating fluid passes through adetector region, different fluid depth passes through the detectorregion. And depending on a particular detection means used, thisdifference in fluid depth can cause a higher “background noise” or aninaccurate reading by the detector. By reducing or eliminating thisfluid oscillation, the “background noise” is reduced and a more accuratereading by the detector can be achieved.

[0237] Preferred devices are constructed by single and multilayer softlithography (MLSL) as detailed in commonly assigned U.S. patentapplication Ser. No. 09/605,520, filed Jun. 27, 2000, which isincorporated herein by reference in its entirety.

[0238] Microfluidic devices of the present invention comprise anintegrated pump which can be electronic, magnetic, mechanical, orpreferably pneumatic pumps. By using a pneumatic pump, microfluidicdevices of the present invention allow more precise control of the fluidflow within the fluid channel. In addition, unlike electro-osmoticdriven fluid flow, pneumatic pump allows the flow of fluids in bothdirections, thereby allowing reversible sorting of materials, asdiscussed in greater detail below. Furthermore, a pneumatic pumpprovides at least 10 times, preferably at least about 20 times, and morepreferably at least about 30 times the fluid flow rate capacity comparedto the capacity of electro-osmotic fluid flow.

[0239] In addition, microfluidic devices of the present invention maycomprise a damper which reduces or eliminates the fluid oscillationwithin the fluid channel. The damper can any device which attenuates thefluid oscillation. For example, the damper can simply be a channel whichis open to the ambient atmosphere and has a thin elastic membranebetween the channel and the fluid flow channel. Preferably, the damperis an encapsulated pocket of fluid medium with a thin elastic membraneabove the fluid flow channel. The fluid medium can be a liquid or,preferably, a gas. The damper is generally located above the fluid flowchannels with a thin membrane, preferably an elastic membrane, betweenthe fluid flow channel and the damper. Typically, there is at least 1damper posterior to the pump in the direction of the fluid flow.Preferably, there is at least 2 dampers, more preferably at least 3dampers and most preferably at least about 5 dampers posterior to thepump.

[0240] The width of damper is at least as wide as the width of flowchannel that is located below the damper. In this manner, the entirecross-section of the flow channel is covered by the damper to ensureattenuation of fluid oscillation across the entire width of the flowchannel. Preferably, the width of damper is at least about 1.1 times thewidth of flow channel, more preferably at least about 1.3 times thewidth of the flow channel, and most preferably at least about 1.5 timesthe width of the flow channel. In this manner, the need for a precisealignment of the damper on top of the flow channel is eliminated.

[0241] The damper is separated from the fluid flow channel by a thinmembrane. Preferably this thin membrane has sufficient elasticity todeflect “upward” when a fluid having a peak of sinusoidal wave-likepasses underneath. In this manner, some of the fluid oscillation energyis absorbed by the damper, thereby reducing the height (i.e., peak) offluid oscillation. Typically, the thickness of the membrane between thedamper and the fluid flow channel depends on a variety of factorsincluding the depth and width of the flow channel, the amount of fluidoscillation produced by the pump and the elasticity (i.e., the material)of the membrane. One of ordinary skill in the art can readily determinethe proper membrane thickness to achieve a desired attenuation of fluidoscillation depending on a desired application and materials used.

[0242] In general, damper structures in accordance with embodiments ofthe present invention feature an energy absorber adjacent to the flowchannel, the energy absorber configured to experience a physical changein response to an oscillation within the flow channel. As describedbelow, the energy absorber can take several forms, including but notlimited to a flexible membrane, a pocket filled with a compressiblefluid, and/or flexible walls of the flow channel itself.

[0243]FIG. 26 shows a cross-sectional view of a first embodiment of adamper structure in accordance with the present invention. Damperstructure 2600 comprises cavity 2602 separated from underlying flowchannel 2604 by membrane 2606 of elastomer layer 2608 in which cavity2602 is formed. Oscillation in pressure 2610 in the fluid flowingthrough underlying flow channel 2604 causes membrane 2606 to flex up anddown, thereby absorbing some of the energy of oscillation and providingfor a more uniform flow of material through channel 2604. The degree towhich damper structure 2600 is capable of absorbing oscillation energyis dictated by a number of factors, including but not limited to, thelength of the flow channel covered by the membrane, the elasticity ofthe membrane, and the compressability of any liquid material present inthe cavity. The longer the membrane and the greater its flexion, thelarger amount of oscillation energy that can be absorbed. Moreover, asstated above, the damping effect can be amplified by positioning aseries of damper structures along the flow channel.

[0244]FIG. 27 shows a cross-sectional view of yet another embodiment ofa damper structure in accordance with the present invention, whereindamper structure 2700 comprises portion 2702 a of flow channel 2702having a larger cross-section, an upper region of portion 2702 beingfilled with air or some other type of fluid 2704. As material 2706flowing through channel 2702 experiences pressure oscillations 2708 andeventually encounters damper 2700, energy is expended as material 2706pushes against fluid 2704. This expenditure of energy serves to reducethe amplitude of energy of oscillation of material in the flow channel.

[0245]FIG. 28 shows a plan view of yet another embodiment of a damperstructure in accordance with the present invention. Damper structure2800 comprises the combination of cavity 2802 overlying and separatedfrom underlying flow channel 2804 by membrane 2806, and constriction2808 in the width of flow channel 2804 positioned downstream ofcavity/membrane combination 2802/2806. In a manner analogous tooperation of a resistance-capacitance (RC) element of an electroniccircuit, cavity/membrane combination 2802/2806 serves as a flowcapacitor while constriction 2808 serves as a flow resistor, resultingin a reduction in amplitude of pressure oscillations 2810 downstream ofdamper structure 2800.

[0246] While the above embodiments have described dampers that arespecifically implemented as separate structures in the architecture of amicrofluidic device, embodiments of the present invention are notlimited to such structures. For example, the elastomer material in whichflow channels are formed may itself serve to absorb pressureoscillations within the flow, independent of the presence of separatemembrane/cavity structures or fluid filled portions of enlarged flowchannels. The damping effect of the elastomer material upon pressureoscillations would depend upon the elasticity of the particularelastomer, and hence its ability to change shape during absorption ofenergy from the oscillating flow.

[0247] Damper structures in accordance with embodiments of the presentinvention function reduce the amplitude of oscillations by absorbingenergy through displacement of a moveable portion of the damperstructure, for example a flexible membrane or a fluid pocket. The damperstructures will generally operate most efficiently to reduce theamplitude of oscillations within a given frequency range. This frequencyrange is related to the speed of displacement and recovery of themoveable portion relative to the oscillation frequency. The speed ofdisplacement and recovery of the moveable element is in turn dictated bysuch factors as material composition, and the design and dimensions of aspecific damper structure.

[0248] Damper structures in accordance with embodiments of the presentinvention may also be utilized to create fluid circulationsubstructures. This is illustrated and described in conjunction withFIGS. 29A-29B, which show cross-sectional views of embodiments ofcirculation structures utilizing damper structures in accordance withthe present invention.

[0249] Fluid circulation system 2900 comprises flow channel 2902 formedin elastomer material 2904 and featuring valves 2904 and 2906 positionedat either end. End valves 2904 and 2906 are actuated, such thatelastomer valve membranes 2904 a and 2906 a project into and block flowchannel 2902, forming sealed flow channel segment 2902 a.

[0250] Pump 2908 and damper 2910 are positioned adjacent to sealed flowsegment 2902 a. Pump 2908 comprises recess 2908 a separated fromunderlying flow channel 2902 by pump membrane 2908 b. Damper 2910comprises cavity 2910 a separated from underlying flow channel 2902 bydamper membrane 2910 b.

[0251] As shown in FIGS. 29A-B, pump 2908 and damper 2910 cooperate topermit a continuous circulation of fluid within sealed segment 2902 a.Specifically, pump 2908 is first actuated such that pump membrane 2908 bdeflects into flow channel 2902, increasing the pressure within sealedsegment 2902 a. In response to this increased pressure, damper membrane2910 b is displaced into cavity 2910 a and fluid within segment 2902 acirculates to occupy the additional space.

[0252] Subsequently, pump 2908 is deactuated such that pump membrane2908 b relaxes to its original position, out of flow channel 2902.Because of the reduced pressure experienced by segment 2902 a as aresult of the deactuation of pump 2908, damper membrane 2910 b alsorelaxes back into its original position, displacing material back intoflow channel 2902. As a result of this action, the material withinsealed segment 2902 a experiences a back flow, and circulation isaccomplished.

[0253] The circulation of material as just described may prove useful ina number of applications. For example, where a mixture comprisingseveral components is being manipulated by a microfluidic apparatus, thecirculation may serve to ensure homogeneity of the mixture. Similarly,where a suspension is being manipulated, the circulating action mayserve to maintain particles in suspension.

[0254] Moreover, certain components of a fluid being manipulated by amicrofluidic device, such as cellular material, may stick to flowchannel sidewalls. Maintenance of a continuous circulation within theflow channels may help prevent loss of material to the channel walls.

[0255] III. Sorting Applications

[0256] In one particular embodiment of the present invention, themicrofluidic device comprises a T-channel for sorting materials (e.g.,cells or large molecules such as peptides, DNA's and other polymers)with fluid flow channel dimensions of about 50 μm×35 μm (width×depth).The width of pressure channels (i.e., pneumatic pump) and the damper is100 μm and 80 μm, respectively. The gap between the flow channel and thedamper (or the pressure channel) is about 5 to 6 μm. In order to producesuch a thin first layer, the MLSL process requires providing a layer ofan elastomer (e.g., by spreading) which is typically thinner than mostother previously disclosed microfluidic devices. For example, when usingGE RTV 615 PDMS silicon rubber, previous microfluidic devices typicallyused 30:1 ratio of 615A:615B at 2000 rpm spin-coating to fabricate thefirst (i.e., bottom) layer of the elastomer and 3:1 ratio of A:B for thesecond elastomer layer. However, it has been found by the presentinventors that the silicon rubber does not cure when the ratio of 30:1is used in fabricating the above described dimensions of fluid flowchannels in the first elastomer layer.

[0257] Moreover, in order to produce a thin first elastomer layer, ahigher spin-coating rate was required. For example, without using anydiluent, GE RTV 615 PDMS silicon rubber A and B components in the ratioof about 20:1 was required at 8000 rpm to produce the first elastomerlayer having about 3.5 μm flow channel depth and about 5-6 μm thicknessbetween the flow channel and the damper (or the pressure channels). WhenSF-96 diluent was used, spin-coating at about 3000 rpm can be used toachieve a similar dimension first elastomer layer.

[0258] During fabrication of a mold, the photoresist is typically etchedusing a mask, developed and heated. Heating of the developed photoresistreshapes trapezoid-shaped “ridges”, which ultimately form the channels,to a smooth rounded ridges and reduces the height of ridges from about20 μm to about 5 μm. This method, however, does not provide channelshaving depth of about 3.5 μm. The present inventors have found that thislimitation can be overcome by treating the developed photoresist withoxygen plasma (e.g., using SPI Plasma Prep II from SPI Supplies aDivision of Structure Probe, Inc., West Chester, Pa.) and heating thephotoresist at a lower heat setting. Unlike previous methods, where ahigher heat setting appear to chemically modify the photoresist, thelower heat setting used in the present invention does not chemicallyalter the photoresist.

[0259] Microfluidic devices of the present invention can be used in avariety of applications such as sorting cells as disclosed in commonlyassigned U.S. patent application Ser. No. 09/325,667 and thecorresponding published PCT Application No. US99/13050, and sortingDNA's as disclosed in commonly assigned U.S. patent application Ser. No.09/499,943, all of which are incorporated herein by reference in theirentirety.

[0260] The actual dimensions of a particular microfluidic device dependon its application. For example, for sorting bacteria which typicallyhave cell size of about 1 μm, the width of fluid flow channel isgenerally in the range of from about 5 μm to about 50 μm and the depthof at least about 5 μm. For sorting mammalian cells which have typicallyhave cell size of about 30 μm, the width of fluid flow channel isgenerally in the range of from about 40 μm to about 60 μm and the depthof at least about 40 μm. For DNA sorting, the dimensions of fluid flowchannels can be significantly less.

[0261] TABLE A below provides a nonexclusive, nonlimiting list ofcandidate sortable entities, their approximate size range, and theapproximate range of flow channel widths of a microfluidic apparatus atthe point of detection of the entity. TABLE A APPROXIMATE SIZEAPPROXIMATE RANGE OF SORTABLE RANGE OF SORT- FLOW CHANNEL WIDTH ATENTITY ABLE ENTITY (μm) DETECTION POINT (μm) bacterial cell 1-10 5-50mammalian  5-100 10-500 cell egg cell  10-1000  10-1000 sperm cell 1-1010-100 DNA strands 0.003-1    0.1-10   proteins 0.01-1    1-10 micelles0.1-100   1-500 viruses 0.05-1    1-10 larvae 600-6500 VARIABLE beads0.01-100   VARIABLE

[0262] The information presented in TABLE A is exemplary in nature, andis intended only as a nonexclusive listing of candidates for sortingutilizing embodiments in accordance with the present invention. Thesorting of other entities, and variation in approximate channel widthsutilized to sort the entities listed above, are possible and would varyaccording to the particular application.

[0263] One particular embodiment of the present invention is shown inFIGS. 30A and 30B, where FIG. 30A is a schematic drawing of themicrofluidic device shown in FIG. 30B. In this embodiment, themicrofluidic device comprises an injection pool 3022, where a fluidcontaining a material can be introduced. The fluid is then pumpedthrough the fluid flow channel 3034 via a pneumatic pump 3010 whichcomprises three pressure channels. By alternately pressurizing the threepressure channels, one can pump the fluid through the fluid flow channel3034 in a similar fashion to a peristaltic pump.

[0264] The fluid exiting the pump oscillates due to actions of the pump.The fluid oscillation amplitude is attenuated by dampers 3014 which islocated above the flow channel 3034, behind the pump 3010 and before adetector (not shown). Initially, the collection valve 3018A is closedand the waste valve 3018B is open to allow the fluid to flow from theinjection pool 3022 through the T-junction 3038 and into the waste pool3030. When a desired material is detected by the detector (not shown),the waste valve 3018B is closed and the collection valve 3018A is openedto allow the material to be collected in the collection pool 3026. Thevalves 3018A and 3018B are interconnected to the detector through acomputer or other automated system to allow opening and closing ofappropriate valves depending on whether a desired material is detectedor not.

[0265] Dimensions of the embodiment shown above in FIGS. 30A and 30B areas follows. The width of the peristaltic pumps is 100 μm. The width ofthe dampers is 80 μm. The width of the switch valves is 30 and 50 μm.The dimensions of the T channel is 50×3.5 μm. The dimensions at theT-junction are 5×3.5 μm. The thickness of the interlayer is 6 μm.

[0266] One such application for the microfluidic device described aboveis in a reverse sorting of a material (e.g., beads, DNA's, peptides orother polymers, or cells) as shown in FIGS. 31A and 31B. In the reversesorting process, the material is allowed to flow towards the waste pool3030 as shown in FIG. 31A. When a desired material is detected by adetector (not shown) the pump (not shown) is reversed until the materialis again detected by the detector. At this point, the waste valve 3018Bis closed and the collection valve 3018A is opened, as shown in FIG.31B, and the flow of material is again reversed to allow the material toflow into the collection pool 3026. After which the collection valve3018A is closed and the waste valve 3018B is opened. This entire processis repeated until a desired amount of materials in the injection (orinput) pool 3022 is “sorted”. The reverse movement of materials in theflow channel as just described can be assisted by changing pressuresapplied directly to flow channel inlets and outlets.

[0267] In one embodiment of the present invention, E. Coli expressingGFP is sorted using the reversible sorting process described above. Asshown in FIGS. 32A and 32B, the cell velocity depends on the frequencyof the pump. Thus, the cell velocity reaches a maximum of about 16mm/sec at about 100 Hz of pump rate. Moreover, as expected, the meanreverse time in FIG. 31B, which represents the time interval betweendetection of E. Coli expressing GFP, reversing the pump, and detectionof the same E. Coli, decreases as the pump frequency is increased.

[0268] In another embodiment of the present invention provides sortingmaterials according to ratio of wavelengths (e.g., from laser inducedfluorescence). For example, by measuring two different fluorescencewavelengths (e.g., λ1 and λ2) and calculating the ratio of λ1 and λ2,one can determine a variety of information regarding the material, suchas the life cycle stage of cells, the stage of evolution of cells, thestrength of enzyme-substrate binding, the strength of drug interactionswith cells, receptors or enzymes, and other useful biological andnon-biological interactions.

[0269] Another embodiment of the present invention provides multipleinterrogation (i.e., observation or detection) of the same material atdifferent time intervals. For example, by closing of the valves 3018A or3018B in FIG. 33C and alternately pumping the fluid to and from theinput well 3022 at a particular intervals, the material can be made toflow to and from the input well 3022 through the detector (not shown).By oscillating this material through the detection window 3040, one canobserve the material at different time intervals. For example, a samplecan be interrogated at 10 Hz pump frequency as shown in FIG. 33A or at75 Hz pump frequency as shown in FIG. 33B. As expected, at a higher pumpfrequency, the material can be observed at shorter intervals. Suchobservation of materials at different time has variety of applicationsincluding monitoring cell developments, enzyme-substrate interactions,affect of drugs on a given cell or enzyme; measuring half-life of agiven material including drugs, compounds, polymers and the like; aswell as other biological applications.

[0270] The performance of a sorter structure in accordance withembodiments of the present invention may be dependent upon the elastomermaterial utilized to fabricate the device. FIG. 34 plots flow velocityversus pump frequency for cell sorters fabricated from differentelastomeric materials, namely General Electric RTV 615 and Dow CorningSylgard 184.

[0271]FIG. 34 shows that the flow velocity of cells through the cellsorters reached a maximum at a pumping frequency of about 50 Hz. Thedecline in flow velocity above this frequency may be attributable toincomplete opening and closing of the valves with each pumping cycle.

[0272] Moreover, different values for maximum pumping rates of the twosorting structures are different. The RTV 615 cell sorter exhibits amaximum pumping rate of about 10,000 μm/sec, while the Sylgard 184 cellsorter exhibits a maximum pumping rate of about 14,000 μm/sec. Maximumflow rates of other elastomeric microfluidic devices in accordance withthe present invention have ranged from about 6000 μm/sec to about 17,000μm/sec, but should be understood as merely exemplary and not limiting tothe scope of the present invention.

[0273]FIG. 34 indicates that the pumping rate of a cell sorter devicemay be controlled by the identity, and hence flexibility, of theparticular elastomer used. In the instant case, based upon the relativeflexibility of RTV 615 and Sylgard 184, the greater the elasticity ofthe elastomer results in a faster rate of pumping.

[0274] Other changes, for example the addition of diluents to theelastomer or the mixing of different ratios of A and B components offluidic layer, may allow even further fine tuning of the pumping rate.Changing the dimensions of the fluidic channel may also allow tuning ofthe pumping rate, as different volumes of fluid in the channels will bemoved with each actuation of the membrane.

[0275] While the sorting device described above utilizes a T-shapedjunction between flow channels, this is not required by the presentinvention. Other types of junctions, including but not limited toY-shaped, or even junctions formed by the intersection of four or moreflow channels, could be utilized for sorting and the device would remainwithin the scope of the present invention.

[0276] Moreover, while only a single sorting structure is illustratedabove, the invention is not limited to this particular configuration. Asorter in accordance with embodiments of the present invention isreadily integratable with other structures on the same microfabricateddevice. For example, embodiments in accordance with the presentinvention may include a series of consecutively-arranged sortingstructures useful for segregating different components of a mixturethrough successive sorting operations.

[0277] In addition, a microfluidic device in accordance with embodimentsof the present invention could also include structures for pre-sortingand post-sorting activities that are in direct fluid communication withthe sorter. Examples of pre- or post-sorting activities that can beintegrated directly into a microfluidic device in accordance with thepresent invention include, but are not limited to, crystallization, celllysis, labeling, staining, filtering, separation, dialysis,chromatography, mixing, reaction, polymerase chain reaction, andincubation. Chambers and other structures for performing theseactivities can be integrated directly onto the microfabricatedelastomeric structure.

[0278] While the present invention has been described herein withreference to particular embodiments thereof, a latitude of modification,various changes and substitutions are intended in the foregoingdisclosure, and it will be appreciated that in some instances somefeatures of the invention will be employed without a corresponding useof other features without departing from the scope of the invention asset forth. Therefore, many modifications may be made to adapt aparticular situation or material to the teachings of the inventionwithout departing from the essential scope and spirit of the presentinvention. It is intended that the invention not be limited to theparticular embodiment disclosed as the best mode contemplated forcarrying out this invention, but that the invention will include allembodiments and equivalents falling within the scope of the claims.

What is claimed is:
 1. A microfluidic device comprising: a flow channel;a pump operatively interconnected to said flow channel for moving afluid in said flow channel; and a damper operatively interconnected tosaid flow channel for reducing the fluid oscillation in said flowchannel.
 2. The microfluidic device of claim 1, further comprising aflow control valve operatively interconnected to said flow channel forclosing and opening said flow channel.
 3. The microfluidic device ofclaim 2, further comprising a T-junction.
 4. The microfluidic device ofclaim 3, wherein said T-junction comprises an injection pool, a wastepool and a collection pool interconnected by said flow channel, andwherein flow channel further comprises said flow control valve proximalto said waste pool and said flow control valve proximal to saidcollection pool, said pump proximal to said injection pool and saiddamper proximal to said injection pool but posterial to said pump. 5.The microfluidic device of claim 4 further comprising a plurality ofsaid dampers.
 6. The microfluidic device of claim 1 wherein the dampercomprises a cavity separated from the flow channel by a flexiblemembrane, the flexible membrane deflectable into the cavity to absorbenergy in response to pressure oscillation within the flow channel,thereby reducing an amplitude of the pressure oscillation.
 7. Themicrofluidic device of claim 6 further comprising a constriction in awidth of the flow channel positioned downstream of the flexiblemembrane.
 8. The microfluidic device of claim 1 wherein the dampercomprises an enlarged portion of the flow channel partially filled witha fluid, the fluid compressible to absorb energy in response to pressureoscillation within the flow channel, thereby reducing an amplitude ofthe pressure oscillation.
 9. The microfluidic device of claim 1 whereinthe damper comprises elastomeric material forming walls of the flowchannel, the elastomeric material deflectable to absorb energy inresponse to pressure oscillation within the flow channel, therebyreducing an amplitude of the pressure oscillation.
 10. A microfluidicsorting device comprising: a first flow channel formed in a first layerof elastomer material, a first end of the first flow channel in fluidcommunication with a collection pool and a second end of the first flowchannel in fluid communication with a waste pool; a second flow channelformed in the first elastomer layer, a first end of the second flowchannel in fluid communication with an injection pool and a second endof the second flow channel in fluid communication with the first flowchannel at a junction; a collection valve adjacent to a first side ofthe junction proximate to the collection pool, the collection valvecomprising a first recess formed in a second elastomer layer overlyingthe first elastomer layer, the first recess separated from the firstflow channel by a first membrane portion of the second elastomer layerdeflectable into the first flow channel; a waste valve adjacent to asecond side of the junction proximate to the waste pool, the waste valvecomprising a second recess formed in the second elastomer layerseparated from the second flow channel by a second membrane portion ofthe second elastomer layer deflectable into the first flow channel; apump adjacent to a third side of the junction proximate to the injectionpool, the pump comprising at least pressure channels formed in thesecond elastomer layer and separated from second flow channel by thirdmembrane portions of the second elastomer layer deflectable into thesecond flow channel; and a detection region positioned between theinjection pool and the junction, one of an open and closed state of thecollection valve and the waste valve determined by an identity of asortable entity detected in the detection region.
 11. The microfluidicdevice of claim 10 further comprising a damper structure adjacent to thesecond flow channel between the pump and the detection region.
 12. Themicrofluidic device of claim 11 wherein the damper comprises a cavityformed in the second elastomer layer and separated from the second flowchannel by a flexible membrane, the flexible membrane deflectable intothe cavity to absorb energy in response to pressure oscillation withinthe second flow channel, thereby reducing an amplitude of the pressureoscillation.
 13. The microfluidic device of claim 12 further comprisinga constriction in a width of the second flow channel between theflexible membrane and the junction.
 14. The microfluidic device of claim11 wherein the damper comprises an enlarged portion of the flow channelpartially filled with a fluid, the fluid compressible to absorb energyin response to pressure oscillation within the flow channel, therebyreducing an amplitude of the pressure oscillation.
 15. The microfluidicdevice of claim 10 wherein the elastomer material forming walls of thefirst flow channel is deflectable to absorb energy in response topressure oscillation within the flow channel, thereby reducing anamplitude of the pressure oscillation.
 16. The microfluidic device ofclaim 10 wherein the junction is T-shaped.
 17. The microfluidic deviceof claim 10 further comprising a second sorter structure positionedbetween the waste valve and the waste pool.
 18. A damper for amicrofluidic device comprising: a flow channel formed in an elastomermaterial; and an energy absorber adjacent to the flow channel andconfigured to absorb an energy of oscillation of a fluid positionedwithin the flow channel.
 19. The damper of claim 18 wherein the energyabsorber comprises a flexible elastomer membrane positioned between theflow channel and a cavity, the flexible membrane deflectable into thecavity to absorb the energy of oscillation in the flow channel.
 20. Thedamper of claim 18 wherein the energy absorber comprises a fluidpositioned within an enlarged portion of the flow channel, the fluidcompressible to absorb the energy of oscillation.
 21. The damper ofclaim 18 wherein the energy absorber comprises elastomer material on theside walls of the flow channel, the elastomer material deflectable toabsorb the energy of oscillation.
 22. The damper of claims 19, 20, or 21further comprising a constriction in a width of the flow channeldownstream of the energy absorber.
 23. A sorting method comprising:deflecting a first elastomer membrane of an elastomer block into a flowchannel to cause a sortable entity to flow into a detection regionpositioned upstream of a junction in the flow channel; interrogating thedetection region to identify the sortable entity within the detectionregion; based upon an identity of the sortable entity, deflecting one ofa second membrane and a third membrane of the elastomer block into oneof a first branch flow channel portion and a second branch flow channelportion located downstream of the junction to cause the sortable entityto flow to one of a collection pool or a waste pool.
 24. The method ofclaim 23 wherein the sortable entity is flowed once through thedetection region.
 25. The method of claim 23 wherein: the sortableentity is initially flowed through the detection region; and then adirection of flow is reversed to flow the sortable entity back into thedetection region.
 26. The method of claim 23 further comprisingdampening an oscillation of energy within the flow channel upstream ofthe junction.
 27. A method for sorting a material using a microfluidicdevice of claim
 4. 28. The method of claim 27 comprising using areversible sorting process.
 29. A method for dampening pressureoscillations in a flow channel comprising providing an energy absorberadjacent to the flow channel, such that the energy absorber experiencesa change in response the pressure oscillations.
 30. The method of claim29 wherein the energy absorber comprises a flexible membrane thatdeflects into a cavity in response to the pressure oscillations.
 31. Themethod of claim 29 wherein the energy absorber comprises a fluid pocketthat experiences compression in response to the pressure oscillations.32. The method of claim 29 wherein the energy absorber comprises anelastomeric flow channel sidewall that experiences deformation inresponse to the pressure oscillations.
 33. The method of claim 29further comprising constricting a width of the flow channel downstreamof the energy absorber.